Drought tolerant plants and related constructs and methods involving genes encoding dtp4 polypeptides

ABSTRACT

Isolated polynucleotides and polypeptides and recombinant DNA constructs useful for conferring stress tolerance are presented herein, along with compositions (such as plants or seeds) comprising these recombinant DNA constructs, and methods utilizing these recombinant DNA constructs. The recombinant DNA construct comprises a polynucleotide operably linked to a promoter that is functional in a plant, wherein said polynucleotide encodes a DTP4 polypeptide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/921,754, filed Dec. 30, 2013, the entire content of which is hereinincorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20141218_BB1672PCT_SequenceListing created on Dec. 18, 2014 and having asize of 1,461 kilobytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

FIELD

The field relates to plant breeding and genetics and, in particular,relates to recombinant DNA constructs useful in plants for conferringtolerance to drought.

BACKGROUND

Abiotic stress is the primary cause of crop loss worldwide, causingaverage yield losses of more than 50% for major crops (Boyer, J. S.(1982) Science 218:443-448; Bray, E. A. et al. (2000) In Biochemistryand Molecular Biology of Plants, Edited by Buchannan, B. B. et al.,Amer. Soc. Plant Biol., pp. 1158-1203). Among the various abioticstresses, drought is the major factor that limits crop productivityworldwide. Exposure of plants to a water-limiting environment duringvarious developmental stages appears to activate various physiologicaland developmental changes. Understanding of the basic biochemical andmolecular mechanism for drought stress perception, transduction andtolerance is a major challenge in biology. Reviews on the molecularmechanisms of abiotic stress responses and the genetic regulatorynetworks of drought stress tolerance have been published (Valliyodan,B., and Nguyen, H. T., (2006) Curr. Opin. Plant Biol. 9:189-195; Wang,W., et al. (2003) Planta 218:1-14); Vinocur, B., and Altman, A. (2005)Curr. Opin. Biotechnol. 16:123-132; Chaves, M. M., and Oliveira, M. M.(2004) J. Exp. Bot. 55:2365-2384; Shinozaki, K., et al. (2003) Curr.Opin. Plant Biol. 6:410-417; Yamaguchi-Shinozaki, K., and Shinozaki, K.(2005) Trends Plant Sci. 10:88-94).

Another abiotic stress that can limit crop yields is low nitrogenstress. The adsorption of nitrogen by plants plays an important role intheir growth (Gallais et al., J. Exp. Bot. 55(396):295-306 (2004)).Plants synthesize amino acids from inorganic nitrogen in theenvironment. Consequently, nitrogen fertilization has been a powerfultool for increasing the yield of cultivated plants, such as maize andsoybean. If the nitrogen assimilation capacity of a plant can beincreased, then increases in plant growth and yield increase are alsoexpected. In summary, plant varieties that have better nitrogen useefficiency (NUE) are desirable.

SUMMARY

The present disclosure includes:

One embodiment of the current disclosure is a plant comprising in itsgenome a recombinant DNA construct comprising a polynucleotide operablylinked to at least one heterologous regulatory element, wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 80% sequence identity, when compared to SEQ ID NO:18, 39, 43, 45,47, 49, 51, 55, 59, 61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117,119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628, and whereinsaid plant exhibits at least one phenotype selected from the groupconsisting of: increased triple stress tolerance, increased droughtstress tolerance, increased nitrogen stress tolerance, increased osmoticstress tolerance, altered ABA response, altered root architecture,increased tiller number, when compared to a control plant not comprisingsaid recombinant DNA construct. In one embodiment said plant exhibits anincrease in yield, biomass, or both, when compared to a control plantnot comprising said recombinant DNA construct. In one embodiment, saidplant exhibits said increase in yield, biomass, or both when compared,under water limiting conditions, to said control plant not comprisingsaid recombinant DNA construct.

One embodiment of the current disclosure also includes seed of theplants disclosed herein, wherein said seed comprises in its genome arecombinant DNA construct comprising a polynucleotide operably linked toat least one heterologous regulatory element, wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 80% sequence identity, when compared to SEQ ID NO:18, 39, 43, 45,47, 49, 51,55, 59, 61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117,119, 121,123, 127, 129, 130, 131,132, 135, 627 or 628, and wherein aplant produced from said seed exhibits an increase in at least onephenotype selected from the group consisting of: drought stresstolerance, triple stress tolerance, osmotic stress tolerance, nitrogenstress tolerance, tiller number, yield and biomass, when compared to acontrol plant not comprising said recombinant DNA construct.

One embodiment of the current disclosure is a method of increasingstress tolerance in a plant, wherein the stress is selected from a groupconsisting of: drought stress, triple stress, nitrogen stress andosmotic stress, the method comprising: (a) introducing into aregenerable plant cell a recombinant DNA construct comprising apolynucleotide operably linked to at least one heterologous regulatorysequence, wherein the polynucleotide encodes a polypeptide having anamino acid sequence of at least 80% sequence identity, when compared toSEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64, 65, 66, 95, 97, 101,103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135,627 or 628; (b) regenerating a transgenic plant from the regenerableplant cell of (a), wherein the transgenic plant comprises in its genomethe recombinant DNA construct; and (c) obtaining a progeny plant derivedfrom the transgenic plant of (b), wherein said progeny plant comprisesin its genome the recombinant DNA construct and exhibits increasedtolerance to at least one stress selected from the group consisting ofdrought stress, triple stress, nitrogen stress and osmotic stress, whencompared to a control plant not comprising the recombinant DNAconstruct.

The current disclosure also encompasses a method of selecting forincreased stress tolerance in a plant, wherein the stress is selectedfrom a group consisting of: drought stress, triple stress, nitrogenstress and osmotic stress, the method comprising: (a) obtaining atransgenic plant, wherein the transgenic plant comprises in its genome arecombinant DNA construct comprising a polynucleotide operably linked toat least one heterologous regulatory element, wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 80% sequence identity, when compared to SEQ ID NO:18, 39, 43, 45,47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101, 103, 107, 111, 113,117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628; (b)growing the transgenic plant of part (a) under conditions wherein thepolynucleotide is expressed; and (c) selecting the transgenic plant ofpart (b) with increased stress tolerance, wherein the stress is selectedfrom the group consisting of: drought stress, triple stress, nitrogenstress and osmotic stress, when compared to a control plant notcomprising the recombinant DNA construct.

One embodiment of the current disclosure is a method of selecting for analteration of yield, biomass, or both in a plant, comprising: (a)obtaining a transgenic plant, wherein the transgenic plant comprises inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory element, wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 80% sequence identity, when compared to SEQ ID NO:18, 39, 43, 45,47, 49, 51, 55, 59, 61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117,119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628; (b) growing thetransgenic plant of part (a) under conditions wherein the polynucleotideis expressed; and (c) selecting the transgenic plant of part (b) thatexhibits an alteration of yield, biomass or both when compared to acontrol plant not comprising the recombinant DNA construct. In oneembodiment, said selecting step (c) comprises determining whether thetransgenic plant of (b) exhibits an alteration of yield, biomass or bothwhen compared, under water limiting conditions, to a control plant notcomprising the recombinant DNA construct. In one embodiment, saidalteration is an increase.

The current disclosure also encompasses an isolated polynucleotidecomprising: (a) a nucleotide sequence encoding a polypeptide with stresstolerance activity, wherein the stress is selected from a groupconsisting of drought stress, triple stress, nitrogen stress and osmoticstress, and wherein the polypeptide has an amino acid sequence of atleast 95% sequence identity when compared to SEQ ID NO:18, 39, 43, 45,47, 49, 51,55, 59, 61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117,119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628; or (b) the fullcomplement of the nucleotide sequence of (a). The amino acid sequence ofthe polypeptide comprises SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59,61, 64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123,127, 129, 130, 131, 132, 135, 627 or 628. In one embodiment, thenucleotide sequence comprises SEQ ID NO:16, 17, 19, 38, 42, 44, 46, 48,50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112, 116, 118, 120or 122.

The current disclosure also encompasses a plant or seed comprising arecombinant DNA construct, wherein the recombinant DNA constructcomprises any of the polynucleotides disclosed herein, wherein thepolynucleotide is operably linked to at least one heterologousregulatory sequence.

In another embodiment, a plant comprising in its genome an endogenouspolynucleotide operably linked to at least one heterologous regulatoryelement, wherein said endogenous polynucleotide encodes a polypeptidehaving an amino acid sequence of at least 80% sequence identity, whencompared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61,64, 65, 66,95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131,132, 135, 627 or 628, and wherein said plant exhibits at least onephenotype selected from the group consisting of increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, when compared to acontrol plant not comprising the heterologous regulatory element.

One embodiment is a method of increasing in a crop plant at least onephenotype selected from the group consisting of: triple stresstolerance, drought stress tolerance, nitrogen stress tolerance, osmoticstress tolerance, ABA response, tiller number, yield and biomass, themethod comprising increasing the expression of a carboxylesterase in thecrop plant. In one embodiment, the crop plant is maize. In oneembodiment, the carboxylesterase has at least 80% sequence identity,when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61, 64,65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129,130, 131, 132, 135, 627 or 628. In one embodiment, the carboxylesterasegives an E-value score of 1E-15 or less when queried using a ProfileHidden Markov Model prepared using SEQ ID NOS:18, 29, 33, 45, 47, 53,55, 61, 64, 65, 77, 78, 101, 103, 105, 107, 111, 115, 131, 132, 135,137, 139, 141, 144, 433, 559 and 604, the query being carried out usingthe hmmsearch algorithm wherein the Z parameter is set to 1 billion.

Another embodiment is a method of making a plant that exhibits at leastone phenotype selected from the group consisting of: increased triplestress tolerance, increased drought stress tolerance, increased nitrogenstress tolerance, increased osmotic stress tolerance, altered ABAresponse, altered root architecture, increased tiller number, increasedyield and increased biomass, when compared to a control plant, themethod comprising the steps of introducing into a plant a recombinantDNA construct comprising a polynucleotide operably linked to at leastone heterologous regulatory element, wherein said polynucleotide encodesa polypeptide having an amino acid sequence of at least 80% sequenceidentity, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59,61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127,129, 130, 131, 132, 135, 627 or 628.

Another embodiment is a method of producing a plant that exhibits atleast one phenotype selected from the group consisting of: increasedtriple stress tolerance, increased drought stress tolerance, increasednitrogen stress tolerance, increased osmotic stress tolerance, alteredABA response, altered root architecture, increased tiller number,increased yield and increased biomass, wherein the method comprisesgrowing a plant from a seed comprising a recombinant DNA construct,wherein the recombinant DNA construct comprises a polynucleotideoperably linked to at least one heterologous regulatory element, whereinthe polynucleotide encodes a polypeptide having an amino acid sequenceof at least 80% sequence identity, when compared to SEQ ID NO:18, 39,43, 45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101, 103, 107, 111,113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628,wherein the plant exhibits at least one phenotype selected from thegroup consisting of: increased triple stress tolerance, increaseddrought stress tolerance, increased nitrogen stress tolerance, increasedosmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number, increased yield and increasedbiomass, when compared to a control plant not comprising the recombinantDNA construct.

Another embodiment is a method of producing a seed, the methodcomprising the following: (a) crossing a first plant with a secondplant, wherein at least one of the first plant and the second plantcomprises a recombinant DNA construct, wherein the recombinant DNAconstruct comprises a polynucleotide operably linked to at least oneheterologous regulatory element, wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 80% sequenceidentity, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59,61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127,129, 130, 131, 132, 135, 627 or 628; and (b) selecting a seed of thecrossing of step (a), wherein the seed comprises the recombinant DNAconstruct. A plant grown from the seed of part (b) exhibits at least onephenotype selected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, increased yield andincreased biomass, when compared to a control plant not comprising therecombinant DNA construct.

In one embodiment, a method of producing oil or a seed by-product, orboth, from a seed, the method comprising extracting oil or a seedby-product, or both, from a seed that comprises a recombinant DNAconstruct, wherein the recombinant DNA construct comprises apolynucleotide operably linked to at least one heterologous regulatoryelement, wherein the polynucleotide encodes a polypeptide having anamino acid sequence of at least 80% sequence identity, when compared toSEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61, 64, 65, 66, 95, 97,101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132,135, 627 or 628. In one embodiment, the seed is obtained from a plantthat comprises the recombinant DNA construct and exhibits at least onephenotype selected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, increased yield andincreased biomass, when compared to a control plant not comprising therecombinant DNA construct. In one embodiment, the oil or the seedby-product, or both, comprises the recombinant DNA construct.

In another embodiment, the present disclosure includes any of themethods of the present disclosure wherein the plant is selected from thegroup consisting of: Arabidopsis, maize, soybean, sunflower, sorghum,canola, wheat, alfalfa, cotton, rice, barley, millet, sugar cane andswitchgrass.

In another embodiment, the present disclosure concerns a recombinant DNAconstruct comprising any of the isolated polynucleotides of the presentdisclosure operably linked to at least one heterologous regulatorysequence, and a cell, a microorganism, a plant, and a seed comprisingthe recombinant DNA construct. The cell may be eukaryotic, e.g., ayeast, insect or plant cell, or prokaryotic, e.g., a bacterial cell.

In another embodiment, a plant comprising in its genome a recombinantDNA construct comprising a polynucleotide operably linked to at leastone heterologous regulatory element, wherein said polynucleotide encodesa polypeptide having an amino acid sequence of at least 95% sequenceidentity, when compared to SEQ ID NO:18, and wherein said plant exhibitsat least one phenotype selected from the group consisting of: increasedtriple stress tolerance, increased drought stress tolerance, increasednitrogen stress tolerance, increased osmotic stress tolerance, alteredABA response, altered root architecture, increased tiller number,increased yield and increased biomass, when compared to a control plantnot comprising said recombinant DNA construct.

In another embodiment, a method of making a plant that exhibits at leastone phenotype selected from the group consisting of: increased triplestress tolerance, increased drought stress tolerance, increased nitrogenstress tolerance, increased osmotic stress tolerance, altered ABAresponse, altered root architecture, increased tiller number, increasedyield and increased biomass, when compared to a control plant, themethod comprising the steps of introducing into a plant a recombinantDNA construct comprising a polynucleotide operably linked to at leastone heterologous regulatory element, wherein said polynucleotide encodesa polypeptide having an amino acid sequence of at least 95% sequenceidentity, when compared to SEQ ID NO:18.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

The disclosure can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application.

FIG. 1A-FIG. 1G show the alignment of the DTP4 polypeptides which weretested in ABA sensitivity assays (SEQ ID NOS:18, 39, 43, 45, 47, 49, 51,55, 59, 61, 64, 65, 66, 95, 97, 99, 101, 103, 107, 111, 113, 117, 119,121,123, 127, 129, 130, 131, 132, 135, 627 and 628). Residues that areidentical to the residue of consensus sequence (SEQ ID NO:630) at agiven position are enclosed in a box. A consensus sequence (SEQ IDNO:630) is presented where a residue is shown if identical in allsequences, otherwise, a period is shown.

FIG. 1C shows the conserved key residues for an oxyanion hole(represented by asterisks), FIG. 1D shows the conserved nucleophileelbow, FIGS. 1D, 1F and 1G also show the catalytic triad of Ser-His-Aspin shaded boxes. These come together in the tertiary structure of thepolypeptide.

FIG. 2 shows the percent sequence identity and the divergence values foreach pair of amino acids sequences of DTP4 polypeptides displayed inFIG. 1A-1G.

FIG. 3 shows the treatment schedule for screening plants with enhanceddrought tolerance.

FIG. 4 shows the percentage germination response of thepBC-yellow-At5g62180 transgenic and wt col-0 Arabidopsis line in anABA-response assay, at 1 μM ABA.

FIG. 5 shows the yield analysis of maize lines transformed with pCV-DTP4encoding the Arabidopsis lead gene At5g62180.

FIG. 6A and FIG. 6B show the % germination, % greenness and % true leafemergence in a 10-day assay, respectively for the wt Arabidopsis plantsand At5g62180 transgenic line (Line ID 64) at different quadconcentrations. 0% quad is indicated as GM (Growth media).

FIG. 7 shows a graph showing % Germination for the wt and At5g62180transgenic line, after 48 h at 60%, 65% and 70% quad concentrations.

FIG. 8 shows the schematic of the ABA-Root assay.

FIG. 9 shows an effect of different ABA concentrations on the wt andAt5g62180 lines.

FIG. 10 shows the yield analysis of maize lines transformed withpCV-DTP4ac encoding the Arabidopsis lead gene At5g62180, in 1^(st) yearfield testing, under drought stress.

FIG. 10A shows the yield analysis in 7 different locations that arecategorized according to the stress experienced in these locations.

FIG. 10B shows the yield analysis across locations, grouped by stresslevels.

FIG. 11 shows the analysis of the agronomic characteristics of maizelines transformed with pCV-DTP4ac encoding the Arabidopsis lead geneAt5g62180.

FIG. 11A shows the analysis of ear height (EARHT) and plant height(PLANTHT) in maize lines transformed with pCV-DTP4ac encoding theArabidopsis lead gene At5g62180.

FIG. 11B shows the analysis of thermal time to shed (TTSHD), rootlodging or stalk lodging in maize lines transformed with pCV-DTP4acencoding the Arabidopsis lead gene At5g62180.

FIG. 12 shows the percentage germination response of the transgenicArabidopsis plants overexpressing some of the DTP4 polypeptidesdisclosed herein, compared with wt col-0 Arabidopsis line in anABA-response assay, at 1 μM ABA (FIG. 12A) and 2 μM ABA (FIG. 12B). FIG.12 C shows the percentage germination response at 1 μM ABA for some moreDTP4 polypeptides, as explained in Table 8.

FIG. 13 shows the percentage green cotyledon response of the transgenicArabidopsis plants overexpressing some of the DTP4 polypeptidesdisclosed herein, compared with wt col-0 Arabidopsis line in anABA-response assay, at 1 μM ABA, as explained in Table 9.

FIG. 14 shows the yield analysis of maize lines transformed withpCV-DTP4ac encoding the Arabidopsis lead gene At5g62180, in 2^(nd) yearfield testing, under drought stress.

FIG. 14A shows the yield analysis in 8 “no stress” locations.

FIG. 14B shows the yield analysis in 5 “medium stress” locations.

FIG. 14C shows the yield analysis in 5 “severe stress” locations.

FIG. 14 D shows the yield analysis across locations, grouped by droughtstress levels, and the last column shows the yield analysis across alllocations, regardless of stress level.

FIG. 15 shows the yield analysis of maize lines transformed withpCV-DTP4ac encoding the Arabidopsis lead gene At5g62180, under lownitrogen stress.

FIG. 16A shows the yield analysis of maize lines transformed withpCV-CXE8ac encoding the DTP4 polypeptide, AT-CXE8 (At2g45600; SEQ IDNO:64), under different drought stress locations.

FIG. 16B shows the yield analysis of maize lines transformed withpCV-CXE8ac encoding the DTP4 polypeptide, AT-CXE8 (At2g45600; SEQ IDNO:64), across locations, grouped by different drought stress levels.

FIG. 17 shows the detection of DTP4 protein in transgenic maize leavesby mass spectrometry, at growth stage V9. Values are means and standarderrors of 4 field plot replications.

FIG. 18 shows the tiller number in maize plants transformed withpCV-DTP4ac encoding the Arabidopsis lead gene AT-DTP4 (At5g62180), underno stress and drought stress conditions, compared to maize plants notcomprising the Arabidopsis gene.

FIG. 19 shows the root and shoot growth response to ABA in maize plantstransformed with pCV-DTP4ac encoding the Arabidopsis lead gene AT-DTP4(At5g62180), under 0 μM and 10 μM ABA. The graphs represent twodifferent experiments done on two different days.

FIG. 20 shows the leaf area in response to triple stress in maize plantstransformed with pCV-DTP4ac encoding the Arabidopsis lead gene AT-DTP4(At5g62180). The graphs represent leaf area 0, 3 and 6 days aftertreatment (DAT).

FIG. 21 shows the percentage germination response to osmotic stress inmaize plants transformed with pCV-DTP4ac encoding the Arabidopsis leadgene AT-DTP4 (At5g62180). The graphs represent two different experimentsdone on two different days.

FIG. 22 shows shoot growth response in maize plants transformed withpCV-DTP4ac encoding the Arabidopsis lead gene AT-DTP4 (At5g62180), inthe tall clear tube assay.

FIG. 23 shows esterase activity of AT-DTP4 fusion protein expressed inE. coli, with p-nitrophenyl acetate as substrate.

FIG. 24 shows the phylogenetic tree showing DTP4 polypeptides.

SEQ ID NO:1 is the nucleotide sequence of the 4×35S enhancer elementfrom the pHSbarENDs2 activation tagging vector.

SEQ ID NO:2 is the nucleotide sequence of the attP1 site.

SEQ ID NO:3 is the nucleotide sequence of the attP2 site.

SEQ ID NO:4 is the nucleotide sequence of the attL1 site.

SEQ ID NO:5 is the nucleotide sequence of the attL2 site.

SEQ ID NO:6 is the nucleotide sequence of the ubiquitin promoter with 5′UTR and first intron from Zea mays.

SEQ ID NO:7 is the nucleotide sequence of the PinII terminator fromSolanum tuberosum.

SEQ ID NO:8 is the nucleotide sequence of the attR1 site.

SEQ ID NO:9 is the nucleotide sequence of the attR2 site.

SEQ ID NO:10 is the nucleotide sequence of the attB1 site.

SEQ ID NO:11 is the nucleotide sequence of the attB2 site.

SEQ ID NO:12 is the nucleotide sequence of the At5g62180-5′attB forwardprimer, containing the attB1 sequence, used to amplify the At5g62180protein-coding region.

SEQ ID NO:13 is the nucleotide sequence of the At5g62180-3′attB reverseprimer, containing the attB2 sequence, used to amplify the At5g62180protein-coding region.

SEQ ID NO:14 is the nucleotide sequence of the VC062 primer, containingthe T3 promoter and attB1 site, useful to amplify cDNA inserts clonedinto a BLUESCRIPT® II SK(+) vector (Stratagene).

SEQ ID NO:15 is the nucleotide sequence of the VC063 primer, containingthe T7 promoter and attB2 site, useful to amplify cDNA inserts clonedinto a BLUESCRIPT® II SK(+) vector (Stratagene).

SEQ ID NO:16 corresponds to NCBI GI No. 30697645, which is the cDNAsequence from locus At5g62180 encoding an Arabidopsis DTP4 polypeptide.

SEQ ID NO:17 corresponds to the CDS sequence from locus At5g62180encoding an Arabidopsis DTP4 polypeptide.

SEQ ID NO:18 corresponds to the amino acid sequence of At5g62180 encodedby SEQ ID NO:17.

SEQ ID NO:19 corresponds to a sequence of At5g62180 with alternativecodons.

Table 1 presents SEQ ID NOs for the nucleotide sequences obtained fromcDNA clones encoding DTP4 polypeptides from Zea mays, Dennstaedtiapunctilobula, Sesbania bispinosa, Artemisia tridentata, Lamiumamplexicaule, 10 Eschscholzia californica, Linum perenne, Delospermanubigenum, Peperomia caperata, Triglochin maritime, Chlorophytumcomosum, Canna×generalis.

The SEQ ID NOs for the corresponding amino acid sequences encoded by thecDNAs are also presented.

Table 2 presents SEQ ID NOs for more DTP4 polypeptides from publicdatabases.

TABLE 1 cDNAs Encoding DTP4 Polypeptides SEQ ID NO: SEQ ID NO: PlantClone Designation* (Nucleotide) (Amino Acid) Corn cfp2n.pk010.p21 20 21Corn cfp2n.pk070.m7 22 23 Corn cfp3n.pk007.i9 24 25 Corn pco524093 26 27Corn Maize_DTP4-1 28 29 Corn Maize_DTP4-2 30 31 Corn Maize_DTP4-3 32 33Dennstaedtia punctilobula ehsf2n.pk140.e11 34 35 Dennstaedtiapunctilobula ehsf2n.pk147.p21 36 37 Sesbania bispinosa sesgr1n.pk117.j1738 39 Sesbania bispinosa sesgr1n.pk129.m19 40 41 Sesbania bispinosasesgr1n.pk062.h8 42 43 Sesbania bispinosa sesgr1n.pk107.c11 44 45Sesbania bispinosa sesgr1n.pk079.h12 46 47 Artemisia tridentataarttr1n.pk125.i16 48 49 Artemisia tridentata arttr1n.pk029.e11 50 51Artemisia tridentata arttr1n.pk222.b19 52 53 Artemisia tridentataarttr1n.pk120.m9 54 55 Lamium amplexicaule hengr1n.pk028.m4 56 57Delosperma nubigenum icegr1n.pk156.e13 58 59 Peperomia caperata (Emeraldpepgr1n.pk128.o15 60 61 ripple Peperomia) Peperomia caperata (Emeraldpepgr1n.pk190.l24 94 95 ripple Peperomia) Peperomia caperata (Emeraldpepgr1n.pk082.c4 96 97 ripple Peperomia) Linum perenne lpgr1n.pk005.f1998 99 Lamium amplexicaule hengr1n.pk014.d12 100 101 Eschscholziacalifornica ecalgr1n.pk137.m22 102 103 Eschscholzia californicaecalgr1n.pk130.b16 104 105 Amaranthus hypochondriacus ahgr1c.pk108.k16106 107 Sesbania bispinosa sesgr1n.pk022.n10_short 108 109 Artemisiatridentata arttr1n.pk193.a17 110 111 Artemisia tridentataarttr1n.pk090.l10 112 113 Abutilon theophrasti abtgr1na.pk050.o13 150151 Abutilon theophrasti abtgr1na.pk056.o14 152 153 Abutilon theophrastiabtgr1na.pk067.p20 154 155 Amaranthus hypochondriacus ahgr1c.pk004.k17156 157 Amaranthus hypochondriacus ahgr1c.pk206.b6 158 159 Amaranthushypochondriacus ahgr1c.pk239.c17 160 161 Amaranthus hypochondriacusahgr1c.pk101.a18 162 163 Amaranthus hypochondriacus ahgr1c.pk101.b2 164165 Amaranthus hypochondriacus ahgr1c.pk108.m2 166 167 Amaranthushypochondriacus ahgr1c.pk200.a3.r 168 169 Amaranthus hypochondriacusahgr1c.pk228.f18 170 171 Artemisia tridentata arttr1n.pk011.m19 172 173Artemisia tridentata arttr1n.pk025.j17 174 175 Artemisia tridentataarttr1n.pk030.b19 176 177 Artemisia tridentata arttr1n.pk042.k20 178 179Artemisia tridentata arttr1n.pk123.i19 180 181 Artemisia tridentataarttr1n.pk183.a10 182 183 Artemisia tridentata arttr1n.pk101.f15 184 185Artemisia tridentata arttr1n.pk195.e16 186 187 Artemisia tridentataarttr1n.pk047.j22 188 189 Artemisia tridentata arttr1n.pk050.l17 190 191Artemisia tridentata arttr1n.pk006.b12.r 192 193 Artemisia tridentataarttr1n.pk085.i10 194 195 Artemisia tridentata arttr1n.pk144.e19 196 197Artemisia tridentata arttr1n.pk147.k17 198 199 Artemisia tridentataarttr1n.pk014.h9 200 201 Artemisia tridentata arttr1n.pk029.d9 202 203Artemisia tridentata arttr1n.pk187.n1 204 205 Artemisia tridentataarttr1n.pk019.g5 206 207 Artemisia tridentata arttr1n.pk027.i2 208 209Artemisia tridentata arttr1n.pk029.e6 210 211 Artemisia tridentataarttr1n.pk029.p23 212 213 Artemisia tridentata arttr1n.pk046.a17 214 215Artemisia tridentata arttr1n.pk138.c10 216 217 Artemisia tridentataarttr1n.pk152.i9 218 219 Artemisia tridentata arttr1n.pk155.a16 220 221Artemisia tridentata arttr1n.pk158.k23 222 223 Artemisia tridentataarttr1n.pk160.h6 224 225 Artemisia tridentata arttr1n.pk165.c21 226 227Artemisia tridentata arttr1n.pk165.h5 228 229 Artemisia tridentataarttr1n.pk197.d11 230 231 Artemisia tridentata arttr1n.pk199.d13 232 233Artemisia tridentata arttr1n.pk214.l5 234 235 Artemisia tridentataarttr1n.pk218.l1 236 237 Artemisia tridentata arttr1n.pk062.b18 238 239Artemisia tridentata arttr1n.pk104.g4 240 241 Artemisia tridentataarttr1n.pk136.n10 242 243 Artemisia tridentata arttr1n.pk136.p12 244 245Artemisia tridentata arttr1n.pk175.o6 246 247 Artemisia tridentataarttr1n.pk185.f17 248 249 Artemisia tridentata arttr1n.pk206.d14 250 251Artemisia tridentata arttr1n.pk212.n16 252 253 Artemisia tridentataarttr1n.pk218.n13 254 255 Artemisia tridentata arttr1n.pk248.n3 256 257Artemisia tridentata arttr1n.pk203.b15 258 259 Canna × generaliscannagr1n306.pk070.m16 260 261 Canna × generalis cannagr1n306.pk021.c13262 263 Chlorophytum comosum ccgr1n308l56.pk005.i7 264 265 Chlorophytumcomosum ccgr1n.pk045.c6 266 267 Chlorophytum comosumccgr1n308l56.pk011.c6 268 269 Delosperma nubigenum icegr1n.pk047.c2 270271 Delosperma nubigenum icegr1n.pk197.c3 272 273 Delosperma nubigenumicegr1n.pk213.k16 274 275 Delosperma nubigenum icegr1n.pk014.l3.r 276277 Delosperma nubigenum icegr1n.pk116.d7 278 279 Delosperma nubigenumicegr1n.pk035.p22.r 280 281 Delosperma nubigenum icegr1n.pk073.g5.r 282283 Delosperma nubigenum icegr1n.pk162.b18 284 285 Delosperma nubigenumicegr1n.pk219.c22 286 287 Dennstaedtia punctilobula ehsf2n.pk203.m17 288289 Dennstaedtia punctilobula ehsf2n.pk123.n16 290 291 Dennstaedtiapunctilobula ehsf2n.pk148.p1 292 293 Dennstaedtia punctilobulaehsf2n.pk124.a11 294 295 Dennstaedtia punctilobula ehsf2n.pk221.a15 296297 Dennstaedtia punctilobula ehsf2n.pk233.n18 298 299 Dennstaedtiapunctilobula ehsf2n.pk049.b14 300 301 Dennstaedtia punctilobulaehsf2n.pk171.m4 302 303 Eschscholzia californica ecalgr1n.pk193.p13.r304 305 Eschscholzia californica ecalgr1n.pk130.g3 306 307 Eschscholziacalifornica ecalgr1n.pk016.p16 308 309 Eschscholzia californicaecalgr1n.pk042.h15 310 311 Eschscholzia californica ecalgr1n.pk128.h17312 313 Eschscholzia californica ecalgr1n.pk132.f19 314 315 Eschscholziacalifornica ecalgr1n.pk008.m5 316 317 Eschscholzia californicaecalgr1n.pk063.d23 318 319 Eschscholzia californica ecalgr1n.pk070.g7320 321 Eschscholzia californica ecalgr1n.pk121.e22 322 323 Eschscholziacalifornica ecalgr1n.pk132.f20 324 325 Eschscholzia californicaecalgr1n.pk140.c5 326 327 Eschscholzia californica ecalgr1n.pk145.e6 328329 Eschscholzia californica ecalgr1n.pk172.m18 330 331 Eschscholziacalifornica ecalgr1n.pk194.e7 332 333 Eschscholzia californicaecalgr1n.pk152.p24 334 335 Eschscholzia californica ecalgr1n.pk007.a21336 337 Eschscholzia californica ecalgr1n.pk028.m20 338 339 Eschscholziacalifornica ecalgr1n.pk049.n17 340 341 Eschscholzia californicaecalgr1n.pk086.l10 342 343 Eschscholzia californica ecalgr1n.pk092.n18.r344 345 Eschscholzia californica ecalgr1n.pk095.l21 346 347 Eschscholziacalifornica ecalgr1n.pk111.h1 348 349 Eschscholzia californicaecalgr1n.pk142.b14 350 351 Eschscholzia californica ecalgr1n.pk169.l22352 353 Eschscholzia californica ecalgr1n.pk192.l15 354 355 Lamiumamplexicaule hengr1n.pk056.e14 356 357 Lamium amplexicaulehengr1n.pk015.c10 358 359 Lamium amplexicaule hengr1n.pk019.g3 360 361Lamium amplexicaule hengr1n.pk169.h24 362 363 Lamium amplexicaulehengr1n.pk019.a8 364 365 Lamium amplexicaule hengr1n.pk042.e4 366 367Lamium amplexicaule hengr1n.pk106.i3 368 369 Lamium amplexicaulehengr1n.pk183.g9 370 371 Lamium amplexicaule hengr1n.pk006.e14 372 373Lamium amplexicaule hengr1n.pk139.k22.r 374 375 Lamium amplexicaulehengr1n.pk205.e4 376 377 Lamium amplexicaule hengr1n.pk083.p6.r 378 379Lamium amplexicaule hengr1n.pk099.i9 380 381 Lamium amplexicaulehengr1n.pk132.n2 382 383 Lamium amplexicaule hengr1n.pk166.h13 384 385Lamium amplexicaule hengr1n.pk191.p1 386 387 Lamium amplexicaulehengr1n.pk252.o11 388 389 Lamium amplexicaule hengr1n.pk007.p2 390 391Lamium amplexicaule hengr1n.pk121.a23 392 393 Lamium amplexicaulehengr1n.pk062.j19 394 395 Lamium amplexicaule hengr1n.pk104.j11 396 397Lamium amplexicaule hengr1n.pk124.a20 398 399 Lamium amplexicaulehengr1n.pk182.c11 400 401 Lamium amplexicaule hengr1n.pk252.b18 402 403Linum perenne lpgr1n.pk122.d12 404 405 Linum perenne lpgr1n.pk049.d20406 407 Linum perenne lpgr1n.pk023.c23.r 408 409 Linum perennelpgr1n.pk008.f18 410 411 Linum perenne lpgr1n.pk085.m11 412 413 Linumperenne lpgr1n.pk102.p22 414 415 Linum perenne lpgr1n.pk055.f13.r 416417 Linum perenne lpgr1n.pk059.i18.r 418 419 Linum perennelpgr1n.pk074.m24.r 420 421 Linum perenne lpgr1n.pk016.a14 422 423 Linumperenne lpgr1n.pk030.p21 424 425 Linum perenne lpgr1n.pk035.j14 426 427Linum perenne lpgr1n.pk060.a17 428 429 Peperomia caperatapepgr1n.pk053.k21 430 431 Peperomia caperata pepgr1n.pk070.b11 432 433Peperomia caperata pepgr1n.pk098.f11 434 435 Peperomia caperatapepgr1n.pk048.n2 436 437 Peperomia caperata pepgr1n.pk240.d2 438 439Peperomia caperata pepgr1n.pk075.j19 440 441 Peperomia caperatapepgr1n.pk143.g17 442 443 Peperomia caperata pepgr1n.pk224.n19 444 445Peperomia caperata pepgr1n.pk236.p10 446 447 Sesbania bispinosasesgr1n.pk067.o14 448 449 Sesbania bispinosa sesgr1n.pk069.p21 450 451Sesbania bispinosa sesgr1n.pk140.i18 452 453 Sesbania bispinosasesgr1n.pk119.d14 454 455 Sesbania bispinosa sesgr1n.pk059.f22 456 457Sesbania bispinosa sesgr1n.pk108.j9 458 459 Sesbania bispinosasesgr1n.pk019.p14 460 461 Sesbania bispinosa sesgr1n.pk117.d15 462 463Sesbania bispinosa sesgr1n.pk132.p20 464 465 Sesbania bispinosasesgr1n.pk142.e7 466 467 Sesbania bispinosa sesgr1n.pk151.n5 468 469Sesbania bispinosa sesgr1n.pk154.p5 470 471 Sesbania bispinosasesgr1n.pk172.f15 472 473 Sesbania bispinosa sesgr1n.pk120.c11 474 475Sesbania bispinosa sesgr1n.pk007.h12 476 477 Sesbania bispinosasesgr1n.pk024.h4 478 479 Sesbania bispinosa sesgr1n.pk028.i7 480 481Sesbania bispinosa sesgr1n.pk034.p15 482 483 Sesbania bispinosasesgr1n.pk041.p8 484 485 Sesbania bispinosa sesgr1n.pk080.f8 486 487Sesbania bispinosa sesgr1n.pk083.d4 488 489 Sesbania bispinosasesgr1n.pk126.e15 490 491 Sesbania bispinosa sesgr1n.pk172.d10 492 493Triglochin maritima tmgr2n.pk038.g19 494 495 Triglochin maritimatmgr2n308l56.pk045.l21 496 497 Triglochin maritima tmgr2n.pk042.m4.r 498499 Triglochin maritima tmgr2n.pk009.b15 500 501 Triglochin maritimatmgr2n.pk020.a24 502 503 Triglochin maritima tmgr2n.pk036.i19 504 505Triglochin maritima tmgr2n.pk048.f6 506 507 Triglochin maritimatmgr2n308l56.pk031.p21 508 509 *The “Full-Insert Sequence” (“FIS”) isthe sequence of the entire cDNA insert.

SEQ ID NO:62 is the nucleotide sequence encoding AT-CXE8 polypeptide;corresponding to At2g45600 locus (Arabidopsis thaliana).

SEQ ID NO:63 is the AT-CXE8 nucleotide sequence with alternative codons.

SEQ ID NO:64 is the amino acid sequence corresponding to NCBI GI No.75318485 (AT-CXE8), encoded by the sequence given in SEQ ID NO:62 and63; (Arabidopsis thaliana).

SEQ ID NO:65 is the amino acid sequence corresponding to NCBI GI No.75318486 (AT-CXE9), encoded by the locus At2g45610.1 (Arabidopsisthaliana).

SEQ ID NO:66 is the amino acid sequence corresponding to NCBI GI No.75335430 (AT-CXE18), encoded by the locus At5g23530.1 (Arabidopsisthaliana).

SEQ ID NO:67 is the amino acid sequence corresponding to the locusLOC_Os08g43430.1, a rice (japonica) predicted protein from the MichiganState University Rice Genome Annotation Project Osa1 release 6.

SEQ ID NO:68 is the amino acid sequence corresponding to the locusLOC_Os03g14730.1, a rice (japonica) predicted protein from the MichiganState University Rice Genome Annotation Project Osa1 release 6.

SEQ ID NO:69 is the amino acid sequence corresponding to the locusLOC_Os07g44890.1, a rice (japonica) predicted protein from the MichiganState University Rice Genome Annotation Project Osa1 release 6.

SEQ ID NO:70 is the amino acid sequence corresponding to the locusLOC_Os07g44860.1, a rice (japonica) predicted protein from the MichiganState University Rice Genome Annotation Project Osa1 release 6.

SEQ ID NO:71 is the amino acid sequence corresponding to the locusLOC_Os07g44910.1, a rice (japonica) predicted protein from the MichiganState University Rice Genome Annotation Project Osa1 release 6.

SEQ ID NO:72 is the amino acid sequence corresponding to Sb07g025010.1,a sorghum (Sorghum bicolor) predicted protein from the Sorghum JGIgenomic sequence version 1.4 from the US Department of energy JointGenome Institute.

SEQ ID NO:73 is the amino acid sequence corresponding to Sb01g040930.1,a sorghum (Sorghum bicolor) predicted protein from the Sorghum JGIgenomic sequence version 1.4 from the US Department of energy JointGenome Institute.

SEQ ID NO:74 is the amino acid sequence corresponding toGlyma20g29190.1, a soybean (Glycine max) predicted protein frompredicted coding sequences from Soybean JGI Glyma1.01 genomic sequencefrom the US Department of energy Joint Genome Institute.

SEQ ID NO:75 is the amino acid sequence corresponding toGlyma20g29200.1, a soybean (Glycine max) predicted protein frompredicted coding sequences from Soybean JGI Glyma1.01 genomic sequencefrom the US Department of energy Joint Genome Institute.

SEQ ID NO:76 is the amino acid sequence corresponding toGlyma16g32560.1, a soybean (Glycine max) predicted protein frompredicted coding sequences from Soybean JGI Glyma1.01 genomic sequencefrom the US Department of energy Joint Genome Institute.

SEQ ID NO:77 is the amino acid sequence corresponding toGlyma07g09040.1, a soybean (Glycine max) predicted protein frompredicted coding sequences from Soybean JGI Glyma1.01 genomic sequencefrom the US Department of energy Joint Genome Institute.

SEQ ID NO:78 is the amino acid sequence corresponding toGlyma07g09030.1, a soybean (Glycine max) predicted protein frompredicted coding sequences from Soybean JGI Glyma1.01 genomic sequencefrom the US Department of energy Joint Genome Institute.

SEQ ID NO:79 is the amino acid sequence corresponding toGlyma03g02330.1, a soybean (Glycine max) predicted protein frompredicted coding sequences from Soybean JGI Glyma1.01 genomic sequencefrom the US Department of energy Joint Genome Institute.

SEQ ID NO:80 is the amino acid sequence corresponding toGlyma09g27500.1, a soybean (Glycine max) predicted protein frompredicted coding sequences from Soybean JGI Glyma1.01 genomic sequencefrom the US Department of energy Joint Genome Institute.

SEQ ID NO:81 the amino acid sequence presented in SEQ ID NO:12 of U.S.Pat. No. 7,915,050 (Arabidopsis thaliana).

SEQ ID NO:82 is the amino acid sequence corresponding to NCBI GI No.194704970 (Zea mays).

SEQ ID NO:83 the amino acid sequence presented in SEQ ID NO:260345 of USPatent Publication No. US20120216318 (Zea mays).

SEQ ID NO:84 is the amino acid sequence corresponding to NCBI GI No.195636334 (Zea mays).

SEQ ID NO:85 the amino acid sequence presented in SEQ ID NO:331675 of USPatent Publication No. US20120216318.

SEQ ID NO:86 is the amino acid sequence corresponding to NCBI GI No.194707422 (Zea mays).

SEQ ID NO:87 the amino acid sequence presented in SEQ ID NO:7332 of U.S.Pat. No. 8,343,764 (Zea mays).

SEQ ID NO:88 is the amino acid sequence corresponding to NCBI GI No.223948401 (Zea mays).

SEQ ID NO:89 the amino acid sequence presented in SEQ ID NO:16159 ofU.S. Pat. No. 7,569,389 (Zea mays).

SEQ ID NO:90 is the amino acid sequence corresponding to NCBI GI No.23495723 (Oryza sativa).

SEQ ID NO:91 the amino acid sequence presented in SEQ ID NO:50819 of USPatent Publication No. US20120017292 (Zea mays).

SEQ ID NO:92 is the amino acid sequence corresponding to NCBI GI No.215768720 (Oryza sativa).

SEQ ID NO:93 the amino acid sequence presented in SEQ ID NO:10044 ofU.S. Pat. No. 8,362,325 (Sorghum bicolor).

SEQ ID NO:114 is the nucleotide sequence of a DTP4 polypeptide fromCarica papaya.

SEQ ID NO:115 is the amino acid sequence of a polypeptide, encoded bythe nucleotide sequence presented in SEQ ID NO:114 (Carica papaya).

SEQ ID NO:116 is the nucleotide sequence of a polypeptide from Eutremasalsugineum.

SEQ ID NO:117 is the amino acid sequence of a polypeptide, encoded bythe nucleotide sequence presented in SEQ ID NO:116 (Eutremasalsugineum).

SEQ ID NO:118 is the nucleotide sequence of an assembled contig fromBrassica napus and Brassica oleracea sequences(Bn-Bo).

SEQ ID NO:119 is the amino acid sequence of a polypeptide, encoded bythe nucleotide sequence presented in SEQ ID NO:118.

SEQ ID NO:120 is the nucleotide sequence of an assembled contig fromBrassica napus and Brassica oleracea sequences (Bole-someBnap).

SEQ ID NO:121 is the amino acid sequence of a polypeptide, encoded bythe nucleotide sequence presented in SEQ ID NO:120.

SEQ ID NO:122 is the nucleotide sequence of an assembled contig of ESTsfrom Brassica napus.

SEQ ID NO:123 is the amino acid sequence of a polypeptide, encoded bythe nucleotide sequence presented in SEQ ID NO:122.

SEQ ID NO:124 is the nucleotide sequence of an assembled contig of ESTsfrom Citrus sinensis and Citrus clementina.

SEQ ID NO:125 is the amino acid sequence of a DTP4 polypeptide fromCitrus sinensis and Citrus clementina.

SEQ ID NO:126 is the amino acid sequence of a DTP4 polypeptide fromRaphanus sativus.

SEQ ID NO:127 is the amino acid sequence of a DTP4 polypeptide fromArabidopsis lyrata.

SEQ ID NO:128 is the amino acid sequence of a DTP4 polypeptide fromOlimarabidopsis pumila.

SEQ ID NO:129 is the amino acid sequence of a DTP4 polypeptide fromCapsella rubella.

SEQ ID NO:130 is the amino acid sequence of a DTP4 polypeptide fromCapsella rubella.

SEQ ID NO:131 is the amino acid sequence of a DTP4 polypeptide fromBrassica rapa subsp. pekinensis.

SEQ ID NO:132 is the amino acid sequence of a DTP4 polypeptide fromBrassica rapa subsp. pekinensis.

SEQ ID NO:133 is the amino acid sequence of a DTP4 polypeptide fromPrunus persica.

SEQ ID NOS:134 and 135 are the amino acid sequences of 2 DTP4 homologsfrom Vitis vinifera.

SEQ ID NO:136 is the nucleotide sequence of a Vitis vinifera DTP4polypeptide named GSVIVT01027568001 (unique_1).

SEQ ID NO:137 is the amino acid sequence of the DTP4 polypeptidesequence of a Vitis vinifera DTP4 polypeptide (GSVIVT01027568001;unique_1).

SEQ ID NO:138 is the nucleotide sequence of a Vitis vinifera DTP4homolog named GSVIVT01027566001 (unique_2).

SEQ ID NO:139 is the amino acid sequence of the DTP4 polypeptidesequence of a Vitis vinifera DTP4 polypeptide (GSVIVT01027566001;unique_2).

SEQ ID NO:140 is the nucleotide sequence of a Vitis vinifera DTP4homolog named GSVIVT01027569001 (unique_3).

SEQ ID NO:141 is the amino acid sequence of the DTP4 polypeptidesequence of a Vitis vinifera DTP4 polypeptide (GSVIVT01027569001;unique_3).

SEQ ID NOS:142-149 are the amino acid sequences of DTP4 polypeptidesfrom Populus trichocarpa.

SEQ ID NO:627 is the amino acid sequence encoded by the locus At1g49660(AT-CXE5) (Arabidopsis thaliana).

SEQ ID NO:628 is the amino acid sequence encoded by the locus At5g16080(AT-CXE17) (Arabidopsis thaliana).

SEQ ID NO:629 is the sequence of the fusion protein of AT-DTP4overexpressed in E. coli.

SEQ ID NO:630 is the consensus sequence obtained from the alignment ofsequences given in FIG. 1

TABLE 2 DTP4 polypeptides SEQ ID NO: Plant Clone Designation* (AminoAcid) Arabidopsis halleri Araha.2214s0019.1.p 510 Arabidopsis lyrataD7LCK9_Al 511 Arabidopsis thaliana AT3G05120.1_At_CXE10 512 Arabidopsisthaliana AT3G63010.1_At_CXE14 513 Arabidopsis thalianaAT5G27320.1_At_CXE19 514 Arabidopsis thaliana At5g06570_AtCXE15 515Arabidopsis thaliana At1g68620_AtCXE6 516 Boechera strictaBostr.25993s0214.1.p 517 Boechera stricta Bostr.26833s0018.1.p 518Boechera stricta Bostr.26675s0205.1.p 519 Brachypodium distachyonBradi1g67930.1_BRADI 520 Brachypodium distachyon Bradi3g42207.1_BRADI521 Brassica rapa Brara.E00516.1.p 522 Brassica rapa Brara.I00681.1.p523 Brassica rapa Brara.B03796.1.p 524 Brassica rapa Brara.B03797.1.p525 Brassica rapa Brara.E01694.1.p 526 Capsella grandifloraCagra.21374s0001.1.p 527 Capsella grandiflora Cagra.4003s0009.1.p 528Capsella grandiflora Cagra.2481s0010.1.p 529 Capsella rubella R0HTV4_Cr530 Capsella rubella Carubv10011237m 531 Capsella rubellaCarubv10011932m 532 Carica papaya Cpap_18.158_PACid_16411302 533 Caricapapaya Cpap_18.159_PACid_16411303 534 Eutrema salsugineumThhalv10011663m 535 Glycine max Glyma07g09030.1 536 Glycine maxGlyma02g17010.1 537 Glycine max Glyma03g30460.1 538 Glycine maxGlyma09g28580.1 539 Glycine max Glyma09g28590.1 540 Glycine maxGlyma10g02790.1 541 Glycine max Glyma10g29910.1 542 Glycine maxGlyma16g33320.1 543 Glycine max Glyma16g33330.1 544 Glycine maxGlyma16g33340.1 545 Glycine max Glyma20g37430.1 546 Glycine maxGlyma02g27090.1 547 Glycine max Glyma03g36380.1 548 Glycine maxGlyma06g46520.1 549 Glycine max Glyma06g46520.2 550 Glycine maxGlyma10g11060.1 551 Glycine max Glyma12g10250.1 552 Glycine maxGlyma19g39030.1 553 Glycine max Glyma08g47930.1 554 Glycine maxGlyma10g42260.1 555 Glycine max Glyma17g31740.1 556 Glycine maxGlyma18g53580.1 557 Glycine max Glyma20g24780.1 558 Gossypium raimondiiGorai.007G093200.1 559 Gossypium raimondii Gorai.008G282100.1 560 Oryzasativa LOC_Os05g33730.1 561 Oryza sativa LOC_Os06g20200.1 562 Oryzasativa LOC_Os07g41590.1 563 Oryza sativa LOC_Os07g44850.1 564 Oryzasativa LOC_Os07g44900.1 565 Oryza sativa LOC_Os11g13570.1 566 Oryzasativa LOC_Os11g13630.1 567 Oryza sativa LOC_Os11g13670.1 568 Oryzasativa LOC_Os01g06060.1_OsCXE4 569 Oryza sativa LOC_Os01g06210.1_OsCXE2570 Oryza sativa LOC_Os01g06220.1_OsCXE1 571 Oryza sativaLOC_Os03g57640.1 572 Oryza sativa LOC_Os07g06830.1 573 Oryza sativaLOC_Os07g06840.1 574 Oryza sativa LOC_Os07g06850.1 575 Oryza sativaLOC_Os07g06860.1 576 Oryza sativa LOC_Os07g06880.1 577 Oryza sativaLOC_Os03g15270.1 578 Sorghum bicolor Sb02g038880.1 579 Sorghum bicolorSb02g041000.1 580 Sorghum bicolor Sb02g041040.1 581 Sorghum bicolorSb02g041050.1 582 Sorghum bicolor Sb05g007270.1 583 Sorghum bicolorSb05g007290.1 584 Sorghum bicolor Sb09g020080.1 585 Sorghum bicolorSb09g020080.2 586 Sorghum bicolor Sb01g005720.1 587 Sorghum bicolorSb02g003560.1 588 Sorghum bicolor Sb02g003570.1 589 Sorghum bicolorSb02g003580.1 590 Sorghum bicolor Sb02g003600.1 591 Sorghum bicolorSb02g003610.1 592 Sorghum bicolor Sb02g003620.1 593 Sorghum bicolorSb02g003630.1 594 Sorghum bicolor Sb02g020810.1 595 Sorghum bicolorSb03g005560.1 596 Sorghum bicolor Sb03g005570.1 597 Sorghum bicolorSb03g005580.1 598 Sorghum bicolor Sb03g005590.1 599 Sorghum bicolorSb01g040580.1 600 Eutrema salsugineum Thhalv10001557m_PACid_20189097 601Eutrema salsugineum Thhalv10001767m_PACid_20188989 602 Theobroma cacaoThecc1EG005469t1 603 Theobroma cacao Thecc1EG015038t1_edit 604 Theobromacacao Thecc1EG032452t1 605 Vitis vinifera GSVIVT01027566001 606 Vitisvinifera GSVIVT01027569001 607 Zea mays Maize_DTP4-4 608 Zea maysMaize_DTP4-5 609 Zea mays Maize_DTP4-6 610 Zea mays Maize_DTP4-7 611 Zeamays Maize_DTP4-8 612 Zea mays Maize_DTP4-9 613 Zea mays Maize_DTP4-10614 Zea mays Maize_DTP4-11 615 Zea mays Maize_DTP4-12 616 Zea maysMaize_DTP4-13 617 Zea mays Maize_DTP4-14 618 Zea mays Maize_DTP4-15 619Zea mays Maize_DTP4-16 620 Zea mays Maize_DTP4-17 621 Zea maysMaize_DTP4-18 622 Zea mays Maize_DTP4-19 623 Zea mays Maize_DTP4-20 624Zea mays Maize_DTP4-21 625 Zea mays Maize_DTP4-22 626

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825.

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

The term “AT-DTP4” generally refers to an Arabidopsis thaliana proteinthat is encoded by the Arabidopsis thaliana locus At5g62180. The terms“AT-DTP4”, “AT-CXE20”, “AT-carboxyesterase” and “AT-carboxylesterase 20”are used interchangeably herein. “DTP4 polypeptide” refers herein to theAT-DTP4 polypeptide and its homologs or orthologs from other organisms.The terms Zm-DTP4 and Gm-DTP4 refer respectively to Zea mays and Glycinemax proteins that are homologous to AT-DTP4.

The term DTP4 polypeptide as described herein refers to any of the DTP4polypeptides given in Table 1 and Table 2 in the specification.

The term DTP4 polypeptide also encompasses a polypeptide wherein thepolypeptide gives an E-value score of 1E-15 or less when queried using aProfile Hidden Markov Model prepared using SEQ ID NOS:18, 29, 33, 45,47, 53, 55, 61, 64, 65, 77, 78, 101, 103, 105, 107, 111, 115, 131, 132,135, 137, 139, 141, 144, 433, 559 and 604, the query being carried outusing the hmmsearch algorithm wherein the Z parameter is set to 1billion. The term DTP4 polypeptide also refers herein to a polypeptidewherein the polypeptide gives an E-value score of 1E-15 or less whenqueried using the Profile Hidden Markov Model given in Table 18.

Nakajima et al (Plant Journal (2006) 46, 880-889) have shown thatAT-DTP4 polypeptide sequence has homology to gibberellin receptors, noGA binding capability was detectable in recombinant AT-DTP4polypeptides.

Based on phylogenetic analysis, Marshall et al have identified theprotein encoded by At5g62180 as a carboxylesterase (CXE). By RT-PCRexpression analysis, at-cxe20 was shown to be expressed in almost allArabidopsis tissues (Marshall et al J Mol Evol (2003) 57:487-500).

The main feature of carboxylesterases (or carboxyesterases) is theconserved catalytic triad. The active site is made up of a serine(surrounded by the conserved consensus sequence G-X-S-X-G), a glutamate(or less frequently an aspartate), and a histidine (Marshall et al J MolEvol (2003) 57:487-500). These residues are dispersed throughout theprimary amino acid sequence but come together in the tertiary structureto form a charge relay system, creating a nucleophilic serine that canattack the substrate. Another structural motif of importance is theoxyanion hole, which is involved in stabilizing the substrate-enzymeintermediate during hydrolysis. The oxyanion hole is created by threesmall amino acids: two glycine residues typically located betweenb-strand 3 and a-helix 1 and the third located immediately following thecatalytic serine residue (Marshall et al J Mol Evol (2003) 57:487-500).

The AT-CXE20 polypeptide has a conserved “nucleophile elbow” (G×S×G)with a unique conformation to activate the nucleophile residue S166, theconserved catalytic triad at S166-H302-D272 and the “oxyanion hole” withthe conserved residues G88-G89-G90 for stabilizing the negativelycharged transition state.

Some of these conserved sites and residues are shown in the alignmentfigures (FIG. 1).

Esterases that are part of the alpha/beta hydrolase_3 fold (Pfam domainPF07859) form the group of hydrolases that are expected to providedrought tolerance and/or increased yield for crop plants.

The terms “monocot” and “monocotyledonous plant” are usedinterchangeably herein. A monocot of the current disclosure includes theGramineae.

The terms “dicot” and “dicotyledonous plant” are used interchangeablyherein. A dicot of the current disclosure includes the followingfamilies: Brassicaceae, Leguminosae, and Solanaceae.

The terms “full complement” and “full-length complement” are usedinterchangeably herein, and refer to a complement of a given nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from acDNA library and therefore is a sequence which has been transcribed. AnEST is typically obtained by a single sequencing pass of a cDNA insert.The sequence of an entire cDNA insert is termed the “Full-InsertSequence” (“FIS”). A “Contig” sequence is a sequence assembled from twoor more sequences that can be selected from, but not limited to, thegroup consisting of an EST, FIS and PCR sequence. A sequence encoding anentire or functional protein is termed a “Complete Gene Sequence”(“CGS”) and can be derived from an FIS or a contig.

A “trait” generally refers to a physiological, morphological,biochemical, or physical characteristic of a plant or a particular plantmaterial or cell. In some instances, this characteristic is visible tothe human eye, such as seed or plant size, or can be measured bybiochemical techniques, such as detecting the protein, starch, or oilcontent of seed or leaves, or by observation of a metabolic orphysiological process, e.g. by measuring tolerance to water deprivationor particular salt or sugar concentrations, or by the observation of theexpression level of a gene or genes, or by agricultural observationssuch as osmotic stress tolerance or yield. The term “trait” is usedinterchangeably with the term “phenotype” herein.

“Agronomic characteristic” is a measurable parameter including but notlimited to, abiotic stress tolerance, greenness, yield, growth rate,biomass, fresh weight at maturation, dry weight at maturation, fruityield, seed yield, total plant nitrogen content, fruit nitrogen content,seed nitrogen content, nitrogen content in a vegetative tissue, totalplant free amino acid content, fruit free amino acid content, seed freeamino acid content, free amino acid content in a vegetative tissue,total plant protein content, fruit protein content, seed proteincontent, protein content in a vegetative tissue, drought tolerance,nitrogen uptake, root lodging, harvest index, stalk lodging, plantheight, ear height, ear length, leaf number, tiller number, growth rate,first pollen shed time, first silk emergence time, anthesis silkinginterval (ASI), stalk diameter, root architecture, staygreen, relativewater content, water use, water use efficiency; dry weight of eithermain plant, tillers, primary ear, main plant and tillers or cobs; rowsof kernels, total plant weight·kernel weight, kernel number, salttolerance, chlorophyll content, flavonol content, number of yellowleaves, early seedling vigor and seedling emergence under lowtemperature stress. These agronomic characteristics maybe measured atany stage of the plant development. One or more of these agronomiccharacteristics may be measured under stress or non-stress conditions,and may show alteration on overexpression of the recombinant constructsdisclosed herein.

“Tiller number” herein refers to the average number of tillers on aplant. A tiller is defined as a secondary shoot that has developed andhas a tassel capable of shedding pollen (U.S. Pat. No. 7,723,584).

Tillers are grain-bearing branches in monocotyledonous plants. Thenumber of tillers per plant is a key factor that determines yield in themany major cereal crops, such as rice and wheat, therefore by increasingtiller number, there is a potential for increasing the yield of majorcereal crops like rice, wheat, and barley.

Abiotic stress may be at least one condition selected from the groupconsisting of: drought, water deprivation, flood, high light intensity,high temperature, low temperature, salinity, etiolation, defoliation,heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrientexcess, UV irradiation, atmospheric pollution (e.g., ozone) and exposureto chemicals (e.g., paraquat) that induce production of reactive oxygenspecies (ROS).

“Increased stress tolerance” of a plant is measured relative to areference or control plant, and is a trait of the plant to survive understress conditions over prolonged periods of time, without exhibiting thesame degree of physiological or physical deterioration relative to thereference or control plant grown under similar stress conditions.

A plant with “increased stress tolerance” can exhibit increasedtolerance to one or more different stress conditions.

“Stress tolerance activity” of a polypeptide indicates thatover-expression of the polypeptide in a transgenic plant confersincreased stress tolerance to the transgenic plant relative to areference or control plant.

A polypeptide with a certain activity, such as a polypeptide with one ormore than one activity selected from the group consisting of: increasedtriple stress tolerance, increased drought stress tolerance, increasednitrogen stress tolerance, increased osmotic stress tolerance, alteredABA response, altered root architecture, increased tiller number;indicates that overexpression of the polypeptide in a plant confers thecorresponding phenotype to the plant relative to a reference or controlplant. For example, a plant overexpressing a polypeptide with “alteredABA response activity”, would exhibit the phenotype of “altered ABAresponse”, when compared to a control or reference plant.

Increased biomass can be measured, for example, as an increase in plantheight, plant total leaf area, plant fresh weight, plant dry weight orplant seed yield, as compared with control plants.

The ability to increase the biomass or size of a plant would haveseveral important commercial applications. Crop species may be generatedthat produce larger cultivars, generating higher yield in, for example,plants in which the vegetative portion of the plant is useful as food,biofuel or both.

Increased leaf size may be of particular interest. Increasing leafbiomass can be used to increase production of plant-derivedpharmaceutical or industrial products. An increase in total plantphotosynthesis is typically achieved by increasing leaf area of theplant. Additional photosynthetic capacity may be used to increase theyield derived from particular plant tissue, including the leaves, roots,fruits or seed, or permit the growth of a plant under decreased lightintensity or under high light intensity.

Modification of the biomass of another tissue, such as root tissue, maybe useful to improve a plants ability to grow under harsh environmentalconditions, including drought or nutrient deprivation, because largerroots may better reach water or nutrients or take up water or nutrients.

For some ornamental plants, the ability to provide larger varietieswould be highly desirable. For many plants, including fruit-bearingtrees, trees that are used for lumber production, or trees and shrubsthat serve as view or wind screens, increased stature provides improvedbenefits in the forms of greater yield or improved screening.

The growth and emergence of maize silks has a considerable importance inthe determination of yield under drought (Fuad-Hassan et al. 2008 PlantCell Environ. 31:1349-1360). When soil water deficit occurs beforeflowering, silk emergence out of the husks is delayed while anthesis islargely unaffected, resulting in an increased anthesis-silking interval(ASI) (Edmeades et al. 2000 Physiology and Modeling Kernel set in Maize(eds M. E. Westgate & K. Boote; CSSA (Crop Science Society of America)Special Publication No. 29. Madison, Wis.: CSSA, 43-73). Selection forreduced ASI has been used successfully to increase drought tolerance ofmaize (Edmeades et al. 1993 Crop Science 33: 1029-1035; Bolanos &Edmeades 1996 Field Crops Research 48:65-80; Bruce et al. 2002 J. Exp.Botany 53:13-25).

Terms used herein to describe thermal time include “growing degree days”(GDD), “growing degree units” (GDU) and “heat units” (HU).

“Transgenic” generally refers to any cell, cell line, callus, tissue,plant part or plant, the genome of which has been altered by thepresence of a heterologous nucleic acid, such as a recombinant DNAconstruct, including those initial transgenic events as well as thosecreated by sexual crosses or asexual propagation from the initialtransgenic event. The term “transgenic” as used herein does notencompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,plant propagules, seeds and plant cells and progeny of same. Plant cellsinclude, without limitation, cells from seeds, suspension cultures,embryos, meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores.

“Propagule” includes all products of meiosis and mitosis able topropagate a new plant, including but not limited to, seeds, spores andparts of a plant that serve as a means of vegetative reproduction, suchas corms, tubers, offsets, or runners. Propagule also includes graftswhere one portion of a plant is grafted to another portion of adifferent plant (even one of a different species) to create a livingorganism. Propagule also includes all plants and seeds produced bycloning or by bringing together meiotic products, or allowing meioticproducts to come together to form an embryo or fertilized egg (naturallyor with human intervention).

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. For example, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

The commercial development of genetically improved germplasm has alsoadvanced to the stage of introducing multiple traits into crop plants,often referred to as a gene stacking approach. In this approach,multiple genes conferring different characteristics of interest can beintroduced into a plant. Gene stacking can be accomplished by many meansincluding but not limited to co-transformation, retransformation, andcrossing lines with different transgenes.

“Transgenic plant” also includes reference to plants which comprise morethan one heterologous polynucleotide within their genome. Eachheterologous polynucleotide may confer a different trait to thetransgenic plant.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably and is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” generally refers to the RNA that is withoutintrons and that can be translated into protein by the cell.

“cDNA” generally refers to a DNA that is complementary to andsynthesized from a mRNA template using the enzyme reverse transcriptase.The cDNA can be single-stranded or converted into the double-strandedform using the Klenow fragment of DNA polymerase I.

“Coding region” generally refers to the portion of a messenger RNA (orthe corresponding portion of another nucleic acid molecule such as a DNAmolecule) which encodes a protein or polypeptide. “Non-coding region”generally refers to all portions of a messenger RNA or other nucleicacid molecule that are not a coding region, including but not limitedto, for example, the promoter region, 5′ untranslated region (“UTR”), 3′UTR, intron and terminator. The terms “coding region” and “codingsequence” are used interchangeably herein. The terms “non-coding region”and “non-coding sequence” are used interchangeably herein.

“Mature” protein generally refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or pro-peptides present inthe primary translation product have been removed.

“Precursor” protein generally refers to the primary product oftranslation of mRNA; i.e., with pre- and pro-peptides still present.Pre- and pro-peptides may be and are not limited to intracellularlocalization signals.

“Isolated” generally refers to materials, such as nucleic acid moleculesand/or proteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

As used herein the terms non-genomic nucleic acid sequence ornon-genomic nucleic acid molecule generally refer to a nucleic acidmolecule that has one or more change in the nucleic acid sequencecompared to a native or genomic nucleic acid sequence. In someembodiments the change to a native or genomic nucleic acid moleculeincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; codon optimization of the nucleicacid sequence for expression in plants; changes in the nucleic acidsequence to introduce at least one amino acid substitution, insertion,deletion and/or addition compared to the native or genomic sequence;removal of one or more intron associated with a genomic nucleic acidsequence; insertion of one or more heterologous introns; deletion of oneor more upstream or downstream regulatory regions associated with agenomic nucleic acid sequence; insertion of one or more heterologousupstream or downstream regulatory regions; deletion of the 5′ and/or 3′untranslated region associated with a genomic nucleic acid sequence; andinsertion of a heterologous 5′ and/or 3′ untranslated region.

“Recombinant” generally refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” generally refers to a combination of nucleicacid fragments that are not normally found together in nature.Accordingly, a recombinant DNA construct may comprise regulatorysequences and coding sequences that are derived from different sources,or regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different than that normally found innature. The terms “recombinant DNA construct” and “recombinantconstruct” are used interchangeably herein.

The terms “entry clone” and “entry vector” are used interchangeablyherein.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to,promoters, translation leader sequences, introns, and polyadenylationrecognition sequences. The terms “regulatory sequence” and “regulatoryelement” are used interchangeably herein.

“Promoter” generally refers to a nucleic acid fragment capable ofcontrolling transcription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably, and refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” generally refers to a promoterwhose activity is determined by developmental events.

“Operably linked” generally refers to the association of nucleic acidfragments in a single fragment so that the function of one is regulatedby the other. For example, a promoter is operably linked with a nucleicacid fragment when it is capable of regulating the transcription of thatnucleic acid fragment.

“Expression” generally refers to the production of a functional product.For example, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein generally refers to both stabletransformation and transient transformation.

“Stable transformation” generally refers to the introduction of anucleic acid fragment into a genome of a host organism resulting ingenetically stable inheritance. Once stably transformed, the nucleicacid fragment is stably integrated in the genome of the host organismand any subsequent generation.

“Transient transformation” generally refers to the introduction of anucleic acid fragment into the nucleus, or DNA-containing organelle, ofa host organism resulting in gene expression without genetically stableinheritance.

“Allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome. When the alleles present at a given locus on apair of homologous chromosomes in a diploid plant are the same thatplant is homozygous at that locus. If the alleles present at a givenlocus on a pair of homologous chromosomes in a diploid plant differ thatplant is heterozygous at that locus. If a transgene is present on one ofa pair of homologous chromosomes in a diploid plant that plant ishemizygous at that locus.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made (Lee et al. (2008) Plant Cell 20:1603-1622). The terms“chloroplast transit peptide” and “plastid transit peptide” are usedinterchangeably herein. “Chloroplast transit sequence” generally refersto a nucleotide sequence that encodes a chloroplast transit peptide. A“signal peptide” is an amino acid sequence which is translated inconjunction with a protein and directs the protein to the secretorysystem (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol.42:21-53). If the protein is to be directed to a vacuole, a vacuolartargeting signal (supra) can further be added, or if to the endoplasmicreticulum, an endoplasmic reticulum retention signal (supra) may beadded. If the protein is to be directed to the nucleus, any signalpeptide present should be removed and instead a nuclear localizationsignal included (Raikhel (1992) Plant Phys. 100:1627-1632). A“mitochondrial signal peptide” is an amino acid sequence which directs aprecursor protein into the mitochondria (Zhang and Glaser (2002) TrendsPlant Sci 7:14-21).

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the Megalign® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal V method of alignment(Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters(GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments and calculation of percent identity of protein sequencesusing the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAPPENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of thesequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Alternatively, the Clustal W method of alignment may be used. TheClustal W method of alignment (described by Higgins and Sharp, CABIOS.5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191(1992)) can be found in the MegAlign™ v6.1 program of the LASERGENE®bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Defaultparameters for multiple alignment correspond to GAP PENALTY=10, GAPLENGTH PENALTY=0.2, Delay Divergent Sequences=30%, DNA TransitionWeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.For pairwise alignments the default parameters areAlignment=Slow-Accurate, Gap Penalty=10.0, Gap Length=0.10, ProteinWeight Matrix=Gonnet 250 and DNA Weight Matrix=IUB. After alignment ofthe sequences using the Clustal W program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table in the same program.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

Complete sequences and figures for vectors described herein (e.g.,pHSbarENDs2, pDONRM/Zeo, pDONRM221, pBC-yellow, PHP27840, PHP23236,PHP10523, PHP23235 and PHP28647) are given in PCT Publication No.WO/2012/058528, the contents of which are herein incorporated byreference.

Turning now to the embodiments:

Embodiments include isolated polynucleotides and polypeptides,recombinant DNA constructs useful for conferring drought tolerance,compositions (such as plants or seeds) comprising these recombinant DNAconstructs, and methods utilizing these recombinant DNA constructs.

Isolated Polynucleotides and Polypeptides:

The present disclosure includes the following isolated polynucleotidesand polypeptides:

An isolated polynucleotide comprising: (i) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V or Clustal W method of alignment, when compared to SEQ IDNO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101, 103,107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or628, and combinations thereof; or (ii) a full complement of the nucleicacid sequence of (i), wherein the full complement and the nucleic acidsequence of (i) consist of the same number of nucleotides and are 100%complementary. Any of the foregoing isolated polynucleotides may beutilized in any recombinant DNA constructs (including suppression DNAconstructs) of the present disclosure. The polypeptide is preferably aDTP4 polypeptide. The polypeptide preferably has stress toleranceactivity, wherein the stress is selected from the group consisting ofdrought stress, triple stress, osmotic stress and nitrogen stress. Thepolypeptide may also have at least one activity selected from the groupconsisting of: carboxylesterase, increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number.

An isolated polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V or Clustal W method of alignment, when compared to SEQ IDNO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64, 65, 66, 95, 97, 101, 103,107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or628, and combinations thereof. The polypeptide is preferably a DTP4polypeptide. The polypeptide preferably has stress tolerance activity,wherein the stress is selected from the group consisting of droughtstress, triple stress, nitrogen stress and osmotic stress. Thepolypeptide may also have at least one activity selected from the groupconsisting of carboxylesterase, increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number.

An isolated polynucleotide comprising (i) a nucleic acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V or Clustal W method of alignment, when comparedto SEQ ID NO:16, 17, 19, 38, 42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94,96, 100, 102, 106, 110, 112, 116, 118, 120 or 122, and combinationsthereof; or (ii) a full complement of the nucleic acid sequence of (i).Any of the foregoing isolated polynucleotides may be utilized in anyrecombinant DNA constructs (including suppression DNA constructs) of thepresent disclosure. The isolated polynucleotide preferably encodes aDTP4 polypeptide. The polypeptide preferably has stress toleranceactivity, wherein the stress is selected from the group consisting ofdrought stress, triple stress, osmotic stress and nitrogen stress. Thepolypeptide may also have at least one activity selected from the groupconsisting of: carboxylesterase, increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number.

An isolated polynucleotide comprising a nucleotide sequence, wherein thenucleotide sequence is hybridizable under stringent conditions with aDNA molecule comprising the full complement of SEQ ID NO:16, 17, 19, 38,42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112,116, 118, 120 or 122. The isolated polynucleotide preferably encodes aDTP4 polypeptide. The polypeptide preferably has stress toleranceactivity, wherein the stress is selected from the group consisting ofdrought stress, triple stress, osmotic stress and nitrogen stress.

An isolated polynucleotide comprising a nucleotide sequence, wherein thenucleotide sequence is derived from SEQ ID NO:16, 17, 19, 38, 42, 44,46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112, 116,118, 120 or 122 by alteration of one or more nucleotides by at least onemethod selected from the group consisting of: deletion, substitution,addition and insertion. The isolated polynucleotide preferably encodes aDTP4 polypeptide. The polypeptide preferably has stress toleranceactivity, wherein the stress is selected from the group consisting ofdrought stress, triple stress, osmotic stress and nitrogen stress. Thepolypeptide may also have at least one activity selected from the groupconsisting of: carboxylesterase, increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number.

An isolated polynucleotide comprising a nucleotide sequence, wherein thenucleotide sequence corresponds to an allele of SEQ ID NO:16, 17, 19,38, 42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110,112, 116, 118, 120 or 122.

In any of the preceding embodiments, the DTP4 polypeptide can be any ofthe DTP4 polypeptide given in Table 1 and Table 2.

In any of the preceding embodiments, the DTP4 polypeptide may be encodedby any of the nucleotide sequences given in Table 1 and Table 2.

It is understood, as those skilled in the art will appreciate, that thedisclosure encompasses more than the specific exemplary sequences.Alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not affectthe functional properties of the encoded polypeptide, are well known inthe art. For example, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another lesshydrophobic residue, such as glycine, or a more hydrophobic residue,such as valine, leucine, or isoleucine. Similarly, changes which resultin substitution of one negatively charged residue for another, such asaspartic acid for glutamic acid, or one positively charged residue foranother, such as lysine for arginine, can also be expected to produce afunctionally equivalent product. Nucleotide changes which result inalteration of the N-terminal and C-terminal portions of the polypeptidemolecule would also not be expected to alter the activity of thepolypeptide. Each of the proposed modifications is well within theroutine skill in the art, as is determination of retention of biologicalactivity of the encoded products.

The protein of the current disclosure may also be a protein whichcomprises an amino acid sequence comprising deletion, substitution,insertion and/or addition of one or more amino acids in an amino acidsequence presented in SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121,123, 127,129, 130, 131,132, 135, 627 or 628. The substitution may beconservative, which means the replacement of a certain amino acidresidue by another residue having similar physical and chemicalcharacteristics. Non-limiting examples of conservative substitutioninclude replacement between aliphatic group-containing amino acidresidues such as lie, Val, Leu or Ala, and replacement between polarresidues such as Lys-Arg, Glu-Asp or Gln-Asn replacement.

Proteins derived by amino acid deletion, substitution, insertion and/oraddition can be prepared when DNAs encoding their wild-type proteins aresubjected to, for example, well-known site-directed mutagenesis (see,e.g., Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982, whichis hereby incorporated by reference in its entirety). As used herein,the term “one or more amino acids” is intended to mean a possible numberof amino acids which may be deleted, substituted, inserted and/or addedby site-directed mutagenesis.

Site-directed mutagenesis may be accomplished, for example, as followsusing a synthetic oligonucleotide primer that is complementary tosingle-stranded phage DNA to be mutated, except for having a specificmismatch (i.e., a desired mutation). Namely, the above syntheticoligonucleotide is used as a primer to cause synthesis of acomplementary strand by phages, and the resulting duplex DNA is thenused to transform host cells. The transformed bacterial culture isplated on agar, whereby plaques are allowed to form fromphage-containing single cells. As a result, in theory, 50% of newcolonies contain phages with the mutation as a single strand, while theremaining 50% have the original sequence. At a temperature which allowshybridization with DNA completely identical to one having the abovedesired mutation, but not with DNA having the original strand, theresulting plaques are allowed to hybridize with a synthetic probelabeled by kinase treatment. Subsequently, plaques hybridized with theprobe are picked up and cultured for collection of their DNA.

Techniques for allowing deletion, substitution, insertion and/oraddition of one or more amino acids in the amino acid sequences ofbiologically active peptides such as enzymes while retaining theiractivity include site-directed mutagenesis mentioned above, as well asother techniques such as those for treating a gene with a mutagen, andthose in which a gene is selectively cleaved to remove, substitute,insert or add a selected nucleotide or nucleotides, and then ligated.

The protein of the present disclosure may also be a protein which isencoded by a nucleic acid comprising a nucleotide sequence comprisingdeletion, substitution, insertion and/or addition of one or morenucleotides in the nucleotide sequence of SEQ ID NO:16, 17, 19, 38, 42,44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112,116, 118, 120 or 122. Nucleotide deletion, substitution, insertionand/or addition may be accomplished by site-directed mutagenesis orother techniques as mentioned above.

The protein of the present disclosure may also be a protein which isencoded by a nucleic acid comprising a nucleotide sequence hybridizableunder stringent conditions with the complementary strand of thenucleotide sequence of SEQ ID NO:16, 17, 19, 38, 42, 44, 46, 48, 50, 54,58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112, 116, 118, 120 or 122.

The term “under stringent conditions” means that two sequences hybridizeunder moderately or highly stringent conditions. More specifically,moderately stringent conditions can be readily determined by thosehaving ordinary skill in the art, e.g., depending on the length of DNA.The basic conditions are set forth by Sambrook et al., MolecularCloning: A Laboratory Manual, third edition, chapters 6 and 7, ColdSpring Harbor Laboratory Press, 2001 and include the use of a prewashingsolution for nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0), hybridization conditions of about 50% formamide, 2×SSC to 6×SSC atabout 40-50° C. (or other similar hybridization solutions, such asStark's solution, in about 50% formamide at about 42° C.) and washingconditions of, for example, about 40-60° C., 0.5-6×SSC, 0.1% SDS.Preferably, moderately stringent conditions include hybridization (andwashing) at about 50° C. and 6×SSC. Highly stringent conditions can alsobe readily determined by those skilled in the art, e.g., depending onthe length of DNA.

Generally, such conditions include hybridization and/or washing athigher temperature and/or lower salt concentration (such ashybridization at about 65° C., 6×SSC to 0.2×SSC, preferably 6×SSC, morepreferably 2×SSC, most preferably 0.2×SSC), compared to the moderatelystringent conditions. For example, highly stringent conditions mayinclude hybridization as defined above, and washing at approximately65-68° C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15 M NaCl, 10 mMNaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is0.15 M NaCl and 15 mM sodium citrate) in the hybridization and washingbuffers; washing is performed for 15 minutes after hybridization iscompleted.

It is also possible to use a commercially available hybridization kitwhich uses no radioactive substance as a probe. Specific examplesinclude hybridization with an ECL direct labeling & detection system(Amersham). Stringent conditions include, for example, hybridization at42° C. for 4 hours using the hybridization buffer included in the kit,which is supplemented with 5% (w/v) Blocking reagent and 0.5 M NaCl, andwashing twice in 0.4% SDS, 0.5×SSC at 55° C. for 20 minutes and once in2×SSC at room temperature for 5 minutes.

DTP4 polypeptides included in the current disclosure are also those thathave an E-value score of 1E-15 or less when queried using a ProfileHidden Markov Model (Profile HMM) prepared using SEQ ID NOS:18, 29, 33,45, 47, 53, 55, 61,64, 65, 77, 78, 101, 103, 105, 107, 111, 115, 131,132, 135, 137, 139, 141, 144, 433, 559 and 604; the query being carriedout using the hmmsearch algorithm wherein the Z parameter is set to 1billion.

In one embodiment, the E-value score can be 1E-15, 1E-25, 1E-35, 1E-45,1E-55, 1E-65, 1E-70, 1E-75, 1E-80 or 1E-85.

The terms “Profile HMMs” or “HMM profile” are used interchangeablyherein as used herein are statistical models of multiple sequencealignments, or even of single sequences. They capture position-specificinformation about how conserved each column of the alignment is, andwhich residues are likely (Krogh et al., 1994, J. Mol. Biol.,235:1501-1531; Eddy, 1998, Curr. Opin. Struct. Biol., 6:361-365.; Durbinet al., Probabilistic Models of Proteins and Nucleic Acids. CambridgeUniversity Press, Cambridge UK. (1998); Eddy, Sean R., March 2010, HMMERUser's Guide Version 3.0, Howard Hughes Medical Institute, Janelia FarmResearch Campus, Ashburn Va., USA; US patent publication No.US20100293118; U.S. Pat. No. 8,623,623).

The term “E-value” or “Expect value (E)” is a parameter which providesthe probability that a match will occur by chance. It provides thestatistical significance of the match to a sequence. The lower theE-value, the more significant the hit. It decreases exponentially as theScore (S) of the match increases.

The Z parameter refers to the ability to set the database size, forpurposes of E-value calculation (Eddy, Sean R., March 2010, HMMER User'sGuide Version 3.0, Howard Hughes Medical Institute, Janelia FarmResearch Campus, Ashburn Va., USA).

Recombinant DNA Constructs and Suppression DNA Constructs:

In one embodiment, the present disclosure includes recombinant DNAconstructs (including suppression DNA constructs).

In one embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one heterologous regulatorysequence (e.g., a promoter functional in a plant), wherein thepolynucleotide comprises (i) a nucleic acid sequence encoding an aminoacid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity, based on the Clustal V or Clustal W method ofalignment, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55,59, 61, 64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123,127, 129, 130, 131, 132, 135, 627 or 628, and combinations thereof; or(ii) a full complement of the nucleic acid sequence of (i). Thepolypeptide may have at least one activity selected from the groupconsisting of carboxylesterase, increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number,

In another embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one heterologous regulatorysequence (e.g., a promoter functional in a plant), wherein saidpolynucleotide comprises (i) a nucleic acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V or Clustal W method of alignment, when compared to SEQ IDNO:16, 17, 19, 38, 42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100,102, 106, 110, 112, 116, 118, 120 or 122, and combinations thereof; or(ii) a full complement of the nucleic acid sequence of (i).

In another embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one heterologous regulatorysequence (e.g., a promoter functional in a plant), wherein saidpolynucleotide encodes a DTP4 polypeptide. The DTP4 polypeptidepreferably has stress tolerance activity, wherein the stress is selectedfrom the group consisting of drought stress, triple stress, osmoticstress and nitrogen stress. The polypeptide may have at least oneactivity selected from the group consisting of carboxylesterase,increased triple stress tolerance, increased drought stress tolerance,increased nitrogen stress tolerance, increased osmotic stress tolerance,altered ABA response, altered root architecture, increased tillernumber,

In any of the embodiments given herein, the DTP4 polypeptide may beselected from any of the polypeptides listed in Table 1 and Table 2.

The DTP4 polypeptide may be from Arabidopsis thaliana, Zea mays, Glycinemax, Glycine tabacina, Glycine soja, Glycine tomentella, Oryza sativa,Brassica napus, Sorghum bicolor, Saccharum officinarum, Triticumaestivum, or any of the plant species disclosed herein.

In one embodiment, a recombinant construct comprises a polynucleotide,wherein the polynucleotide is operably linked to a heterologouspromoter, and encodes a polypeptide with at least one activity selectedfrom the group consisting of: carboxylesterase, increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, wherein thepolypeptide gives an E-value score of 1E-15 or less when queried using aProfile Hidden Markov Model prepared using SEQ ID NOS:18, 29, 33, 45,47, 53, 55, 61, 64, 65, 77, 78, 101, 103, 105, 107, 111, 115, 131, 132,135, 137, 139, 141, 144, 433, 559 and 604, the query being carried outusing the hmmsearch algorithm wherein the Z parameter is set to 1billion.

In another aspect, the present disclosure includes suppression DNAconstructs.

A suppression DNA construct may comprise at least one heterologousregulatory sequence (e.g., a promoter functional in a plant) operablylinked to (a) all or part of: (i) a nucleic acid sequence encoding apolypeptide having an amino acid sequence of at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the ClustalV or Clustal W method of alignment, when compared to SEQ ID NO:18, 39,43, 45, 47, 49, 51,55, 59, 61, 64, 65, 66, 95, 97, 101, 103, 107, 111,113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628, andcombinations thereof, or (ii) a full complement of the nucleic acidsequence of (a)(i); or (b) a region derived from all or part of a sensestrand or antisense strand of a target gene of interest, said regionhaving a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity, based on the Clustal V or Clustal Wmethod of alignment, when compared to said all or part of a sense strandor antisense strand from which said region is derived, and wherein saidtarget gene of interest encodes a DTP4 polypeptide; or (c) all or partof: (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity, based on the Clustal V orClustal W method of alignment, when compared to SEQ ID NO:16, 17, 19,38, 42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110,112, 116, 118, 120 or 122, and combinations thereof, or (ii) a fullcomplement of the nucleic acid sequence of (c)(i). The suppression DNAconstruct may comprise a cosuppression construct, antisense construct,viral-suppression construct, hairpin suppression construct, stem-loopsuppression construct, double-stranded RNA-producing construct, RNAiconstruct, or small RNA construct (e.g., an siRNA construct or an miRNAconstruct).

It is understood, as those skilled in the art will appreciate, that thedisclosure encompasses more than the specific exemplary sequences.Alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not affectthe functional properties of the encoded polypeptide, are well known inthe art. For example, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another lesshydrophobic residue, such as glycine, or a more hydrophobic residue,such as valine, leucine, or isoleucine. Similarly, changes which resultin substitution of one negatively charged residue for another, such asaspartic acid for glutamic acid, or one positively charged residue foranother, such as lysine for arginine, can also be expected to produce afunctionally equivalent product. Nucleotide changes which result inalteration of the N-terminal and C-terminal portions of the polypeptidemolecule would also not be expected to alter the activity of thepolypeptide. Each of the proposed modifications is well within theroutine skill in the art, as is determination of retention of biologicalactivity of the encoded products.

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The terms“suppression”, “suppressing” and “silencing”, used interchangeablyherein, include lowering, reducing, declining, decreasing, inhibiting,eliminating or preventing. “Silencing” or “gene silencing” does notspecify mechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches, and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

A suppression DNA construct may comprise 100, 200, 300, 400, 500, 600,700, 800, 900 or 1000 contiguous nucleotides of the sense strand (orantisense strand) of the gene of interest, and combinations thereof.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

Suppression of gene expression may also be achieved by use of artificialmiRNA precursors, ribozyme constructs and gene disruption. A modifiedplant miRNA precursor may be used, wherein the precursor has beenmodified to replace the miRNA encoding region with a sequence designedto produce a miRNA directed to the nucleotide sequence of interest. Genedisruption may be achieved by use of transposable elements or by use ofchemical agents that cause site-specific mutations.

“Antisense inhibition” generally refers to the production of antisenseRNA transcripts capable of suppressing the expression of the target geneor gene product. “Antisense RNA” generally refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target isolated nucleic acidfragment (U.S. Pat. No. 5,107,065). The complementarity of an antisenseRNA may be with any part of the specific gene transcript, i.e., at the5′ non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence.

“Cosuppression” generally refers to the production of sense RNAtranscripts capable of suppressing the expression of the target gene orgene product. “Sense” RNA generally refers to RNA transcript thatincludes the mRNA and can be translated into protein within a cell or invitro. Cosuppression constructs in plants have been previously designedby focusing on overexpression of a nucleic acid sequence having homologyto a native mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence (seeVaucheret et al., Plant J. 16:651-659 (1998); and Gura, Nature404:804-808 (2000)).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication No.WO 98/36083 published on Aug. 20, 1998).

RNA interference generally refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., Nature 391:806 (1998)). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., Trends Genet.15:358 (1999)).

Small RNAs play an important role in controlling gene expression.Regulation of many developmental processes, including flowering, iscontrolled by small RNAs. It is now possible to engineer changes in geneexpression of plant genes by using transgenic constructs which producesmall RNAs in the plant.

Small RNAs appear to function by base-pairing to complementary RNA orDNA target sequences. When bound to RNA, small RNAs trigger either RNAcleavage or translational inhibition of the target sequence. When boundto DNA target sequences, it is thought that small RNAs can mediate DNAmethylation of the target sequence. The consequence of these events,regardless of the specific mechanism, is that gene expression isinhibited.

MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about 24nucleotides (nt) in length that have been identified in both animals andplants (Lagos-Quintana et al., Science 294:853-858 (2001),Lagos-Quintana et al., Curr. Biol. 12:735-739 (2002); Lau et al.,Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001);Llave et al., Plant Cell 14:1605-1619 (2002); Mourelatos et al., GenesDev. 16:720-728 (2002); Park et al., Curr. Biol. 12:1484-1495 (2002);Reinhart et al., Genes. Dev. 16:1616-1626 (2002)). They are processedfrom longer precursor transcripts that range in size from approximately70 to 200 nt, and these precursor transcripts have the ability to formstable hairpin structures.

MicroRNAs (miRNAs) appear to regulate target genes by binding tocomplementary sequences located in the transcripts produced by thesegenes. It seems likely that miRNAs can enter at least two pathways oftarget gene regulation: (1) translational inhibition; and (2) RNAcleavage. MicroRNAs entering the RNA cleavage pathway are analogous tothe 21-25 nt short interfering RNAs (siRNAs) generated during RNAinterference (RNAi) in animals and posttranscriptional gene silencing(PTGS) in plants, and likely are incorporated into an RNA-inducedsilencing complex (RISC) that is similar or identical to that seen forRNAi.

The terms “miRNA-star sequence” and “miRNA* sequence” are usedinterchangeably herein and they refer to a sequence in the miRNAprecursor that is highly complementary to the miRNA sequence. The miRNAand miRNA* sequences form part of the stem region of the miRNA precursorhairpin structure.

In one embodiment, there is provided a method for the suppression of atarget sequence comprising introducing into a cell a nucleic acidconstruct encoding a miRNA substantially complementary to the target. Insome embodiments the miRNA comprises about 19, 20, 21, 22, 23, 24 or 25nucleotides. In some embodiments the miRNA comprises 21 nucleotides. Insome embodiments the nucleic acid construct encodes the miRNA. In someembodiments the nucleic acid construct encodes a polynucleotideprecursor which may form a double-stranded RNA, or hairpin structurecomprising the miRNA.

In some embodiments, the nucleic acid construct comprises a modifiedendogenous plant miRNA precursor, wherein the precursor has beenmodified to replace the endogenous miRNA encoding region with a sequencedesigned to produce a miRNA directed to the target sequence. The plantmiRNA precursor may be full-length of may comprise a fragment of thefull-length precursor. In some embodiments, the endogenous plant miRNAprecursor is from a dicot or a monocot. In some embodiments theendogenous miRNA precursor is from Arabidopsis, tomato, maize, soybean,sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley,millet, sugar cane or switchgrass.

In some embodiments, the miRNA template, (i.e. the polynucleotideencoding the miRNA), and thereby the miRNA, may comprise some mismatchesrelative to the target sequence. In some embodiments the miRNA templatehas >1 nucleotide mismatch as compared to the target sequence, forexample, the miRNA template can have 1, 2, 3, 4, 5, or more mismatchesas compared to the target sequence. This degree of mismatch may also bedescribed by determining the percent identity of the miRNA template tothe complement of the target sequence. For example, the miRNA templatemay have a percent identity including about at least 70%, 75%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% as compared to the complementof the target sequence.

In some embodiments, the miRNA template, (i.e. the polynucleotideencoding the miRNA) and thereby the miRNA, may comprise some mismatchesrelative to the miRNA-star sequence. In some embodiments the miRNAtemplate has >1 nucleotide mismatch as compared to the miRNA-starsequence, for example, the miRNA template can have 1, 2, 3, 4, 5, ormore mismatches as compared to the miRNA-star sequence. This degree ofmismatch may also be described by determining the percent identity ofthe miRNA template to the complement of the miRNA-star sequence. Forexample, the miRNA template may have a percent identity including aboutat least 70%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%as compared to the complement of the miRNA-star sequence.

Regulatory Sequences:

A recombinant DNA construct (including a suppression DNA construct) ofthe present disclosure may comprise at least one regulatory sequence.

A regulatory sequence may be a promoter.

A number of promoters can be used in recombinant DNA constructs of thepresent disclosure. The promoters can be selected based on the desiredoutcome, and may include constitutive, tissue-specific, inducible, orother promoters for expression in the host organism.

Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”.

High level, constitutive expression of the candidate gene under controlof the 35S or UBI promoter may have pleiotropic effects, althoughcandidate gene efficacy may be estimated when driven by a constitutivepromoter. Use of tissue-specific and/or stress-specific promoters mayeliminate undesirable effects but retain the ability to enhance stresstolerance. This effect has been observed in Arabidopsis (Kasuga et al.(1999) Nature Biotechnol. 17:287-91).

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812(1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), theconstitutive synthetic core promoter SCP1 (International Publication No.03/033651) and the like. Other constitutive promoters include, forexample, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611.

In choosing a promoter to use in the methods of the disclosure, it maybe desirable to use a tissue-specific or developmentally regulatedpromoter.

A tissue-specific or developmentally regulated promoter is a DNAsequence which regulates the expression of a DNA sequence selectively inthe cells/tissues of a plant critical to tassel development, seed set,or both, and limits the expression of such a DNA sequence to the periodof tassel development or seed maturation in the plant. Any identifiablepromoter may be used in the methods of the present disclosure whichcauses the desired temporal and spatial expression.

Promoters which are seed or embryo-specific and may be useful includesoybean Kunitz trypsin inhibitor (Kti3, Jofuku and Goldberg, Plant Cell1:1079-1093 (1989)), patatin (potato tubers) (Rocha-Sosa, M., et al.(1989) EMBO J. 8:23-29), convicilin, vicilin, and legumin (peacotyledons) (Rerie, W. G., et al. (1991) Mol. Gen. Genet. 259:149-157;Newbigin, E. J., et al. (1990) Planta 180:461-470; Higgins, T. J. V., etal. (1988) Plant. Mol. Biol. 11:683-695), zein (maize endosperm)(Schemthaner, J. P., et al. (1988) EMBO J. 7:1249-1255), phaseolin (beancotyledon) (Segupta-Gopalan, C., et al. (1985) Proc. Natl. Acad. Sci.U.S.A. 82:3320-3324), phytohemagglutinin (bean cotyledon) (Voelker, T.et al. (1987) EMBO J. 6:3571-3577), B-conglycinin and glycinin (soybeancotyledon) (Chen, Z-L, et al. (1988) EMBO J. 7:297-302), glutelin (riceendosperm), hordein (barley endosperm) (Marris, C., et al. (1988) PlantMol. Biol. 10:359-366), glutenin and gliadin (wheat endosperm) (Colot,V., et al. (1987) EMBO J. 6:3559-3564), and sporamin (sweet potatotuberous root) (Hattori, T., et al. (1990) Plant Mol. Biol. 14:595-604).Promoters of seed-specific genes operably linked to heterologous codingregions in chimeric gene constructions maintain their temporal andspatial expression pattern in transgenic plants. Such examples includeArabidopsis thaliana 2S seed storage protein gene promoter to expressenkephalin peptides in Arabidopsis and Brassica napus seeds(Vanderkerckhove et al., Bio/Technology 7:L929-932 (1989)), bean lectinand bean beta-phaseolin promoters to express luciferase (Riggs et al.,Plant Sci. 63:47-57 (1989)), and wheat glutenin promoters to expresschloramphenicol acetyl transferase (Colot et al., EMBO J 6:3559-3564(1987)). Endosperm preferred promoters include those described in e.g.,U.S. Pat. No. 8,466,342; U.S. Pat. No. 7,897,841; and U.S. Pat. No.7,847,160.

Inducible promoters selectively express an operably linked DNA sequencein response to the presence of an endogenous or exogenous stimulus, forexample by chemical compounds (chemical inducers) or in response toenvironmental, hormonal, chemical, and/or developmental signals.Inducible or regulated promoters include, for example, promotersregulated by light, heat, stress, flooding or drought, phytohormones,wounding, or chemicals such as ethanol, jasmonate, salicylic acid, orsafeners.

Promoters for use include the following: 1) the stress-inducible RD29Apromoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91); 2) thebarley promoter, B22E; expression of B22E is specific to the pedicel indeveloping maize kernels (“Primary Structure of a Novel Barley GeneDifferentially Expressed in Immature Aleurone Layers”. Klemsdal, S. S.et al., Mol. Gen. Genet. 228(1/2):9-16 (1991)); and 3) maize promoter,Zag2 (“Identification and molecular characterization of ZAG1, the maizehomolog of the Arabidopsis floral homeotic gene AGAMOUS”, Schmidt, R. J.et al., Plant Cell 5(7):729-737 (1993); “Structural characterization,chromosomal localization and phylogenetic evaluation of two pairs ofAGAMOUS-like MADS-box genes from maize”, Theissen et al. Gene156(2):155-166 (1995); NCBI GenBank Accession No. X80206)). Zag2transcripts can be detected 5 days prior to pollination to 7 to 8 daysafter pollination (“DAP”), and directs expression in the carpel ofdeveloping female inflorescences and Ciml which is specific to thenucleus of developing maize kernels. Ciml transcript is detected 4 to 5days before pollination to 6 to 8 DAP. Other useful promoters includeany promoter which can be derived from a gene whose expression ismaternally associated with developing female florets.

Promoters for use also include the following: Zm-GOS2 (maize promoterfor “Gene from Oryza sativa”, US publication number US2012/0110700Sb-RCC (Sorghum promoter for Root Cortical Cell delineating protein,root specific expression), Zm-ADF4 (U.S. Pat. No. 7,902,428; Maizepromoter for Actin Depolymerizing Factor), Zm-FTM1 (U.S. Pat. No.7,842,851; maize promoter for Floral transition MADSs) promoters.

Additional promoters for regulating the expression of the nucleotidesequences in plants are stalk-specific promoters. Such stalk-specificpromoters include the alfalfa S2A promoter (GenBank Accession No.EF030816; Abrahams et al., Plant Mol. Biol. 27:513-528 (1995)) and S2Bpromoter (GenBank Accession No. EF030817) and the like, hereinincorporated by reference.

Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments.

In one embodiment the at least one regulatory element may be anendogenous promoter operably linked to at least one enhancer element;e.g., a 35S, nos or ocs enhancer element.

Promoters for use may include: RIP2, mLIP15, ZmCOR1, Rab17, CaMV 35S,RD29A, B22E, Zag2, SAM synthetase, ubiquitin, CaMV 19S, nos, Adh,sucrose synthase, R-allele, the vascular tissue preferred promoters S2A(Genbank accession number EF030816) and S2B (Genbank accession numberEF030817), and the constitutive promoter GOS2 from Zea mays. Otherpromoters include root preferred promoters, such as the maize NAS2promoter, the maize Cyclo promoter (US 2006/0156439, published Jul. 13,2006), the maize ROOTMET2 promoter (WO05063998, published Jul. 14,2005), the CR1BIO promoter (WO06055487, published May 26, 2006), theCRWAQ81 (WO05035770, published Apr. 21, 2005) and the maize ZRP2.47promoter (NCBI accession number: U38790; GI No. 1063664),

Recombinant DNA constructs of the present disclosure may also includeother regulatory sequences, including but not limited to, translationleader sequences, introns, and polyadenylation recognition sequences. Inanother embodiment of the present disclosure, a recombinant DNAconstruct of the present disclosure further comprises an enhancer orsilencer.

The promoters disclosed herein may be used with their own introns, orwith any heterologous introns to drive expression of the transgene.

An intron sequence can be added to the 5′ untranslated region, theprotein-coding region or the 3′ untranslated region to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold. Buchman and Berg,Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987).

“Transcription terminator”, “termination sequences”, or “terminator”refer to DNA sequences located downstream of a protein-coding sequence,including polyadenylation recognition sequences and other sequencesencoding regulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The use of different 3′ non-coding sequences isexemplified by Ingelbrecht, I. L., et al., Plant Cell 1:671-680 (1989).A polynucleotide sequence with “terminator activity” generally refers toa polynucleotide sequence that, when operably linked to the 3′ end of asecond polynucleotide sequence that is to be expressed, is capable ofterminating transcription from the second polynucleotide sequence andfacilitating efficient 3′ end processing of the messenger RNA resultingin addition of poly A tail. Transcription termination is the process bywhich RNA synthesis by RNA polymerase is stopped and both the processedmessenger RNA and the enzyme are released from the DNA template.

Improper termination of an RNA transcript can affect the stability ofthe RNA, and hence can affect protein expression. Variability oftransgene expression is sometimes attributed to variability oftermination efficiency (Bieri et al (2002) Molecular Breeding 10:107-117).

Examples of terminators for use include, but are not limited to, PinIIterminator, SB-GKAF terminator (U.S. application Ser. No. 14/236,499),Actin terminator, Os-Actin terminator, Ubi terminator, Sb-Ubiterminator, Os-Ubi terminator.

Any plant can be selected for the identification of regulatory sequencesand DTP4 polypeptide genes to be used in recombinant DNA constructs andother compositions (e.g. transgenic plants, seeds and cells) and methodsof the present disclosure. Examples of suitable plants for the isolationof genes and regulatory sequences and for compositions and methods ofthe present disclosure would include but are not limited to alfalfa,apple, apricot, Arabidopsis, artichoke, arugula, asparagus, avocado,banana, barley, beans, beet, blackberry, blueberry, broccoli, brusselssprouts, cabbage, canola, cantaloupe, carrot, cassava, castorbean,cauliflower, celery, cherry, chicory, cilantro, citrus, clementines,clover, coconut, coffee, corn, cotton, cranberry, cucumber, Douglas fir,eggplant, endive, escarole, eucalyptus, fennel, figs, garlic, gourd,grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks, lemon,lime, Loblolly pine, linseed, mango, melon, mushroom, nectarine, nut,oat, oil palm, oil seed rape, okra, olive, onion, orange, an ornamentalplant, palm, papaya, parsley, parsnip, pea, peach, peanut, pear, pepper,persimmon, pine, pineapple, plantain, plum, pomegranate, poplar, potato,pumpkin, quince, radiata pine, radicchio, radish, rapeseed, raspberry,rice, rye, sorghum, Southern pine, soybean, spinach, squash, strawberry,sugarbeet, sugarcane, sunflower, sweet potato, sweetgum, switchgrass,tangerine, tea, tobacco, tomato, triticale, turf, turnip, a vine,watermelon, wheat, yarns, and zucchini.

Compositions:

A composition of the present disclosure includes a transgenicmicroorganism, cell, plant, and seed comprising the recombinant DNAconstruct. The cell may be eukaryotic, e.g., a yeast, insect or plantcell, or prokaryotic, e.g., a bacterial cell.

A composition of the present disclosure is a plant comprising in itsgenome any of the recombinant DNA constructs (including any of thesuppression DNA constructs) of the present disclosure (such as any ofthe constructs discussed above). Compositions also include any progenyof the plant, and any seed obtained from the plant or its progeny,wherein the progeny or seed comprises within its genome the recombinantDNA construct (or suppression DNA construct). Progeny includessubsequent generations obtained by self-pollination or out-crossing of aplant. Progeny also includes hybrids and inbreds.

In hybrid seed propagated crops, mature transgenic plants can beself-pollinated to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced recombinant DNA construct(or suppression DNA construct). These seeds can be grown to produceplants that would exhibit an altered agronomic characteristic (e.g., anincreased agronomic characteristic optionally under stress conditions),or used in a breeding program to produce hybrid seed, which can be grownto produce plants that would exhibit such an altered agronomiccharacteristic. The seeds may be maize seeds. The stress condition maybe selected from the group of drought stress, triple stress and osmoticstress.

The plant may be a monocotyledonous or dicotyledonous plant, forexample, a maize or soybean plant. The plant may also be sunflower,sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, sugarcane or switchgrass. The plant may be a hybrid plant or an inbred plant.

The recombinant DNA construct may be stably integrated into the genomeof the plant.

Particular embodiments include but are not limited to the following:

1. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one heterologous regulatory sequence,wherein said polynucleotide encodes a polypeptide having an amino acidsequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity, based on the Clustal V or Clustal W method ofalignment, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59,61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127,129, 130, 131, 132, 135, 627 or 628, and wherein said plant exhibits atleast one phenotype selected from the group consisting of increasedtriple stress tolerance, increased drought stress tolerance, increasednitrogen stress tolerance, increased osmotic stress tolerance, alteredABA response, altered root architecture, increased tiller number, whencompared to a control plant not comprising said recombinant DNAconstruct. The plant may further exhibit an alteration of at least oneagronomic characteristic when compared to the control plant.

The plant may exhibit alteration of at least one agronomiccharacteristic selected from the group consisting of: abiotic stresstolerance, greenness, yield, growth rate, biomass, fresh weight atmaturation, dry weight at maturation, fruit yield, seed yield, totalplant nitrogen content, fruit nitrogen content, seed nitrogen content,nitrogen content in a vegetative tissue, total plant free amino acidcontent, fruit free amino acid content, seed free amino acid content,free amino acid content in a vegetative tissue, total plant proteincontent, fruit protein content, seed protein content, protein content ina vegetative tissue, drought tolerance, nitrogen uptake, root lodging,harvest index, stalk lodging, plant height, ear height, ear length, leafnumber, tiller number, growth rate, first pollen shed time, first silkemergence time, anthesis silking interval (ASI), stalk diameter, rootarchitecture, staygreen, relative water content, water use, water useefficiency, dry weight of either main plant, tillers, primary ear, mainplant and tillers or cobs; rows of kernels, total plant weight·kernelweight, kernel number, salt tolerance, chlorophyll content, flavonolcontent, number of yellow leaves, early seedling vigor and seedlingemergence under low temperature stress. These agronomic characteristicsmaybe measured at any stage of the plant development. One or more ofthese agronomic characteristics may be measured under stress ornon-stress conditions, and may show alteration on overexpression of therecombinant constructs disclosed herein.

2. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence, wherein saidpolynucleotide encodes a DTP4 polypeptide, and wherein said plantexhibits at least one phenotype selected from the group consisting ofincreased triple stress tolerance, increased drought stress tolerance,increased nitrogen stress tolerance, increased osmotic stress tolerance,altered ABA response, altered root architecture, increased tillernumber, when compared to a control plant not comprising said recombinantDNA construct. The plant may further exhibit an alteration of at leastone agronomic characteristic when compared to the control plant.

3. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence, wherein saidpolynucleotide encodes a DTP4 polypeptide, and wherein said plantexhibits an alteration of at least one agronomic characteristic whencompared to a control plant not comprising said recombinant DNAconstruct.

4. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory element, wherein saidpolynucleotide comprises a nucleotide sequence, wherein the nucleotidesequence is: (a) hybridizable under stringent conditions with a DNAmolecule comprising the full complement of SEQ ID NO:16, 17, 19, 38, 42,44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112,116, 118, 120 or 122; or (b) derived from SEQ ID NO:16, 17, 19, 38, 42,44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112,116, 118, 120 or 122 by alteration of one or more nucleotides by atleast one method selected from the group consisting of: deletion,substitution, addition and insertion; and wherein said plant exhibits atleast one phenotype selected from the group consisting of increasedtriple stress tolerance, increased drought stress tolerance, increasednitrogen stress tolerance, increased osmotic stress tolerance, alteredABA response, altered root architecture, increased tiller number, whencompared to a control plant not comprising said recombinant DNAconstruct. The plant may further exhibit an alteration of at least oneagronomic characteristic when compared to the control plant.

5. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one heterologous regulatory element, whereinsaid polynucleotide encodes a polypeptide having an amino acid sequenceof at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, based on the Clustal V or Clustal W method of alignment, whencompared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64, 65, 66,95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131,132, 135, 627 or 628, and wherein said plant exhibits an alteration ofat least one agronomic characteristic when compared to a control plantnot comprising said recombinant DNA construct.

6. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one heterologous regulatory element, whereinsaid polynucleotide comprises a nucleotide sequence, wherein thenucleotide sequence is: (a) hybridizable under stringent conditions witha DNA molecule comprising the full complement of SEQ ID NO:16, 17, 19,38, 42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110,112, 116, 118, 120 or 122; or (b) derived from SEQ ID NO:16, 17, 19, 38,42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112,116, 118, 120 or 122 by alteration of one or more nucleotides by atleast one method selected from the group consisting of: deletion,substitution, addition and insertion; and wherein said plant exhibits analteration of at least one agronomic characteristic when compared to acontrol plant not comprising said recombinant DNA construct.

7. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one heterologous regulatory sequence,wherein said polynucleotide encodes a polypeptide having an amino acidsequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity, based on the Clustal V or Clustal W method ofalignment, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59,61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127,129, 130, 131, 132, 135, 627 or 628, and wherein said plant exhibits atleast one phenotype selected from the group consisting of increasedtriple stress tolerance, increased drought stress tolerance, increasednitrogen stress tolerance, increased osmotic stress tolerance, alteredABA response, altered root architecture, increased tiller number, whencompared to a control plant not comprising said recombinant DNAconstruct. The plant may further exhibit an increase in yield, biomass,or both when compared to the control plant.

8. A plant (for example, a maize, rice or soybean plant) comprising inits genome a recombinant DNA construct comprising a wherein thepolynucleotide is operably linked to a heterologous promoter, andencodes a polypeptide with at least one activity selected from the groupconsisting of: carboxylesterase, increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number, wherein the polypeptide gives anE-value score of 1E-15 or less when queried using a Profile HiddenMarkov Model prepared using SEQ ID NOS:18, 29, 33, 45, 47, 53, 55, 61,64, 65, 77, 78, 101, 103, 105, 107, 111, 115, 131, 132, 135, 137, 139,141, 144, 433, 559 and 604, the query being carried out using thehmmsearch algorithm wherein the Z parameter is set to 1 billion, andwherein said plant exhibits at least one phenotype selected from thegroup consisting of increased triple stress tolerance, increased droughtstress tolerance, increased nitrogen stress tolerance, increased osmoticstress tolerance, altered ABA response, altered root architecture,increased tiller number, when compared to a control plant not comprisingsaid recombinant DNA construct. The plant may further exhibit anincrease in yield, biomass, or both when compared to the control plant.The polypeptide may give an E-value score of 1E-15, 1E-25, 1E-35, 1E-45,1E-55, 1E-65, 1E-70, 1E-75, 1E-80 and 1E-85.

9. A plant (for example, a maize, rice or soybean plant) comprising inits genome a suppression DNA construct comprising at least oneheterologous regulatory element operably linked to a region derived fromall or part of a sense strand or antisense strand of a target gene ofinterest, said region having a nucleic acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V or Clustal W method of alignment, when compared to saidall or part of a sense strand or antisense strand from which said regionis derived, and wherein said target gene of interest encodes a DTP4polypeptide, and wherein said plant exhibits an alteration of at leastone agronomic characteristic when compared to a control plant notcomprising said suppression DNA construct.

10. A plant (for example, a maize, rice or soybean plant) comprising inits genome a suppression DNA construct comprising at least oneheterologous regulatory element operably linked to all or part of (a) anucleic acid sequence encoding a polypeptide having an amino acidsequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61,64, 65, 66,95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131,132, 135, 627 or 628, or (b) a full complement of the nucleic acidsequence of (a), and wherein said plant exhibits an alteration of atleast one agronomic characteristic when compared to a control plant notcomprising said suppression DNA construct.

11. A plant (for example, a maize, rice or soybean plant) comprising inits genome a polynucleotide (optionally an endogenous polynucleotide)operably linked to at least one heterologous regulatory element, whereinsaid polynucleotide encodes a polypeptide having an amino acid sequenceof at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, based on the Clustal V or Clustal W method of alignment, whencompared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64, 65, 66,95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131,132, 135, 627 or 628, and wherein said plant exhibits at least onephenotype selected from the group consisting of increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number when compared to acontrol plant not comprising the recombinant regulatory element. The atleast one heterologous regulatory element may comprise an enhancersequence or a multimer of identical or different enhancer sequences. Theat least one heterologous regulatory element may comprise one, two,three or four copies of the CaMV 35S enhancer.

12. Any progeny of the plants in the embodiments described herein, anyseeds of the plants in the embodiments described herein, any seeds ofprogeny of the plants in embodiments described herein, and cells fromany of the above plants in embodiments described herein and progenythereof.

In any of the embodiments described herein, the plant may exhibitalteration of at least one agronomic characteristic selected from thegroup consisting of: abiotic stress tolerance, greenness, yield, growthrate, biomass, fresh weight at maturation, dry weight at maturation,fruit yield, seed yield, total plant nitrogen content, fruit nitrogencontent, seed nitrogen content, nitrogen content in a vegetative tissue,total plant free amino acid content, fruit free amino acid content, seedfree amino acid content, free amino acid content in a vegetative tissue,total plant protein content, fruit protein content, seed proteincontent, protein content in a vegetative tissue, drought tolerance,nitrogen uptake, root lodging, harvest index, stalk lodging, plantheight, ear height, ear length, leaf number, tiller number, growth rate,first pollen shed time, first silk emergence time, anthesis silkinginterval (ASI), stalk diameter, root architecture, staygreen, relativewater content, water use, water use efficiency, dry weight of eithermain plant, tillers, primary ear, main plant and tillers or cobs; rowsof kernels, total plant weight·kernel weight, kernel number, salttolerance, chlorophyll content, flavonol content, number of yellowleaves, early seedling vigor and seedling emergence under lowtemperature stress. These agronomic characteristics maybe measured atany stage of the plant development. One or more of these agronomiccharacteristics may be measured under stress or non-stress conditions,and may show alteration on overexpression of the recombinant constructsdisclosed herein.

In any of the embodiments described herein, the DTP4 polypeptide may befrom Arabidopsis thaliana, Zea mays, Glycine max, Glycine tabacina,Glycine soja, Glycine tomentella, Oryza sativa, Brassica napus, Sorghumbicolor, Saccharum officinarum, Triticum aestivum or any other plantspecies disclosed herein.

In any of the embodiments described herein, the recombinant DNAconstruct (or suppression DNA construct) may comprise at least apromoter functional in a plant as a regulatory sequence.

In any of the embodiments described herein or any other embodiments ofthe present disclosure, the alteration of at least one agronomiccharacteristic is either an increase or decrease.

In any of the embodiments described herein, the plant may exhibit thealteration of at least one agronomic characteristic when compared, underat least one stress condition, to a control plant not comprising saidrecombinant DNA construct (or said suppression DNA construct). The atleast one stress condition may be selected from the group consisting ofdrought stress, triple stress, nitrogen stress and osmotic stress.

In one embodiment, “yield” can be measured in many ways, including, forexample, test weight, seed weight, seed number per plant, seed numberper unit area (i.e. seeds, or weight of seeds, per acre), bushels peracre, tonnes per acre, tons per acre, kilo per hectare.

In any of the embodiments described herein, the plant may exhibit lessyield loss relative to the control plants, for example, at least 25%, atleast 20%, at least 15%, at least 10% or at least 5% less yield loss,under water limiting conditions, or would have increased yield, forexample, at least 5%, at least 10%, at least 15%, at least 20% or atleast 25% increased yield, relative to the control plants under waternon-limiting conditions.

In any of the embodiments described herein, the plant may exhibit lessyield loss relative to the control plants, for example, at least 25%, atleast 20%, at least 15%, at least 10% or at least 5% less yield loss,under stress conditions, or would have increased yield, for example, atleast 5%, at least 10%, at least 15%, at least 20% or at least 25%increased yield, relative to the control plants under non-stressconditions. The stress may be selected from the group consisting ofdrought stress, triple stress, nitrogen stress and osmotic stress.

The terms “stress tolerance” or “stress resistance” as used hereingenerally refers to a measure of a plants ability to grow under stressconditions that would detrimentally affect the growth, vigor, yield, andsize, of a “non-tolerant” plant of the same species. Stress tolerantplants grow better under conditions of stress than non-stress tolerantplants of the same species. For example, a plant with increased growthrate, compared to a plant of the same species and/or variety, whensubjected to stress conditions that detrimentally affect the growth ofanother plant of the same species would be said to be stress tolerant. Aplant with “increased stress tolerance” can exhibit increased toleranceto one or more different stress conditions.

“Increased stress tolerance” of a plant is measured relative to areference or control plant, and is a trait of the plant to survive understress conditions over prolonged periods of time, without exhibiting thesame degree of physiological or physical deterioration relative to thereference or control plant grown under similar stress conditions.Typically, when a transgenic plant comprising a recombinant DNAconstruct or suppression DNA construct in its genome exhibits increasedstress tolerance relative to a reference or control plant, the referenceor control plant does not comprise in its genome the recombinant DNAconstruct or suppression DNA construct.

“Drought” generally refers to a decrease in water availability to aplant that, especially when prolonged, can cause damage to the plant orprevent its successful growth (e.g., limiting plant growth or seedyield). “Water limiting conditions” generally refers to a plant growthenvironment where the amount of water is not sufficient to sustainoptimal plant growth and development. The terms “drought” and “waterlimiting conditions” are used interchangeably herein.

“Drought tolerance” is a trait of a plant to survive under droughtconditions over prolonged periods of time without exhibiting substantialphysiological or physical deterioration.

“Drought tolerance activity” of a polypeptide indicates thatover-expression of the polypeptide in a transgenic plant confersincreased drought tolerance to the transgenic plant relative to areference or control plant.

“Increased drought tolerance” of a plant is measured relative to areference or control plant, and is a trait of the plant to survive underdrought conditions over prolonged periods of time, without exhibitingthe same degree of physiological or physical deterioration relative tothe reference or control plant grown under similar drought conditions.Typically, when a transgenic plant comprising a recombinant DNAconstruct or suppression DNA construct in its genome exhibits increaseddrought tolerance relative to a reference or control plant, thereference or control plant does not comprise in its genome therecombinant DNA construct or suppression DNA construct.

“Triple stress” as used herein generally refers to the abiotic stressexerted on the plant by the combination of drought stress, hightemperature stress and high light stress.

The terms “heat stress” and “temperature stress” are usedinterchangeably herein, and are defined as where ambient temperaturesare hot enough for sufficient time that they cause damage to plantfunction or development, which might be reversible or irreversible indamage. “High temperature” can be either “high air temperature” or “highsoil temperature”, “high day temperature” or “high night temperature, ora combination of more than one of these.

In one embodiment of the disclosure, the ambient temperature can be inthe range of 30° C. to 36° C. In one embodiment of the disclosure, theduration for the high temperature stress could be in the range of 1-16hours.

“High light intensity” and “high irradiance” and “light stress” are usedinterchangeably herein, and refer to the stress exerted by subjectingplants to light intensities that are high enough for sufficient timethat they cause photoinhibition damage to the plant.

In one embodiment of the disclosure, the light intensity can be in therange of 250 μE to 450 μE. In one embodiment of the invention, theduration for the high light intensity stress could be in the range of12-16 hours.

“Triple stress tolerance” is a trait of a plant to survive under thecombined stress conditions of drought, high temperature and high lightintensity over prolonged periods of time without exhibiting substantialphysiological or physical deterioration.

“Paraquat” is an herbicide that exerts oxidative stress on the plants.Paraquat, a bipyridylium herbicide, acts by intercepting electrons fromthe electron transport chain at PSI. This reaction results in theproduction of bipyridyl radicals that readily react with dioxygenthereby producing superoxide. Paraquat tolerance in a plant has beenassociated with the scavenging capacity for oxyradicals (Lannelli, M. A.et al (1999) J Exp Botany, Vol. 50, No. 333, pp. 523-532). Paraquatresistant plants have been reported to have higher tolerance to otheroxidative stresses as well.

“Paraquat stress” is defined as stress exerted on the plants bysubjecting them to Paraquat concentrations ranging from 0.03 to 0.3 μM.

Many adverse environmental conditions such as drought, salt stress, anduse of herbicide promote the overproduction of reactive oxygen species(ROS) in plant cells. ROS such as singlet oxygen, superoxide radicals,hydrogen peroxide (H₂O₂), and hydroxyl radicals are believed to be themajor factor responsible for rapid cellular damage due to their highreactivity with membrane lipids, proteins, and DNA (Mittler, R.(2002)Trends Plant Sci Vol. 7 No. 9).

A polypeptide with “triple stress tolerance activity” indicates thatover-expression of the polypeptide in a transgenic plant confersincreased triple stress tolerance to the transgenic plant relative to areference or control plant. A polypeptide with “paraquat stresstolerance activity” indicates that over-expression of the polypeptide ina transgenic plant confers increased Paraquat stress tolerance to thetransgenic plant relative to a reference or control plant.

Typically, when a transgenic plant comprising a recombinant DNAconstruct or suppression DNA construct in its genome exhibits increasedstress tolerance relative to a reference or control plant, the referenceor control plant does not comprise in its genome the recombinant DNAconstruct or suppression DNA construct.

The terms “percentage germination” and “percentage seedling emergence”are used interchangeably herein, and refer to the percentage of seedsthat germinate, when compared to the total number of seeds being tested.

“Germination” as used herein generally refers to the emergence of theradicle.

The term “radicle” as used herein generally refers to the embryonic rootof the plant, and is terminal part of embryonic axis. It grows downwardin the soil, and is the first part of a seedling to emerge from the seedduring the process of germination.

The range of stress and stress response depends on the different plantswhich are used, i.e., it varies for example between a plant such aswheat and a plant such as Arabidopsis.

Osmosis is defined as the movement of water from low soluteconcentration to high solute concentration up a concentration gradient.

“Osmotic pressure” of a solution as defined herein is defined as thepressure exerted by the solute in the system. A solution with higherconcentration of solutes would have higher osmotic pressure. All solutesexhibit osmotic pressure. Osmotic pressure increases as concentration ofthe solute increases.

The osmotic pressure exerted by 250 mM NaCl (sodium chloride) is 1.23MPa (megapascals) (Werner, J. E. et al. (1995) Physiologia Plantarum 93:659-666).

As used herein, the term “osmotic stress” generally refers to any stresswhich is associated with or induced by elevated concentrations ofosmolytes and which result in a perturbation in the osmotic potential ofthe intracellular or extracellular environment of a cell. The term“osmotic stress” as used herein generally refers to stress exerted whenthe osmotic potential of the extracellular environment of the cell,tissue, seed, organ or whole plant is increased and the water potentialis lowered and a substance that blocks water absorption (osmolyte) ispersistently applied to the cell, tissue, seed, organ or whole plant.

With respect to the osmotic stress assay, the term “quad” as used hereinrefers to four components that impart osmotic stress. A “quad assay” or“quad media”, as used herein, would therefore comprise four componentsthat impart osmotic stress, e.g., sodium chloride, sorbitol, mannitoland PEG.

An increase in the osmotic pressure of the media solution would resultin increase in osmotic potential. Examples of conditions that induceosmotic stress include, but are not limited to, salinity, drought, heat,chilling and freezing.

In one embodiment of the disclosure the osmotic pressure of the mediafor subjecting the plants to osmotic stress is from 0.4-1.23 MPa. Inother embodiments of the disclosure, the osmotic pressure of the mediafor subjecting the plants to osmotic stress is 0.4 MPa, 0.5 MPa, 0.6MPa, 0.7 MPa, 0.8 MPa, 0.9 MPa, 1 MPa, 1.1 MPa, 1.2 MPa or 1.23 MPa. Inother embodiments of the disclosure, the osmotic pressure of the mediafor subjecting the plants to osmotic stress is at least 0.4 MPa, 0.5MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa, 0.9 MPa, 1 MPa, 1.1 MPa, 1.2 MPa or 1.23MPa. In another embodiment of the disclosure, the osmotic pressure ofthe media for subjecting the plants to osmotic stress is 1.23 MPa.

“Nitrogen limiting conditions” or “low nitrogen stress” refers toconditions where the amount of total available nitrogen (e.g., fromnitrates, ammonia, or other known sources of nitrogen) is not sufficientto sustain optimal plant growth and development. One skilled in the artwould recognize conditions where total available nitrogen is sufficientto sustain optimal plant growth and development. One skilled in the artwould recognize what constitutes sufficient amounts of total availablenitrogen, and what constitutes soils, media and fertilizer inputs forproviding nitrogen to plants. Nitrogen limiting conditions will varydepending upon a number of factors, including but not limited to, theparticular plant and environmental conditions.

Abscisic acid (ABA), a plant hormone, is known to be involved inimportant plant physiological functions, such as acquisition of stressresponse and tolerance to drought and low temperature, as well as seedmaturation, dormancy, germination etc. (M. Koomneef et al., PlantPhysiol. Biochem. 36:83 (1998); J. Leung & J. Giraudat, Annu. Rev.Plant. Physiol. Plant. Mol. Biol. 49:199 (1998)). Plants subjected toenvironmental stresses such as drought and low temperature are thoughtto acquire the ability to adapt to environmental stresses due to the invivo synthesis of ABA, which causes various changes within the plantcells. A number of genes have been identified that are induced by ABA.This suggests that ABA-induced tolerance to adverse environmentalconditions is a complex multigenic event.

The terms “altered ABA response” and “altered ABA sensitivity” are usedinterchangeably herein, and, as used herein, by these terms it is meantthat a plant or plant part exhibits an altered ABA induced response,when compared to a control plant, and includes both hypersensitivity andhyposensitivity to ABA.

“Hypersensitivity” or “enhanced response” of a plant to ABA means thatthe plant exhibits ABA induced phenotype at lower concentration of ABAthan the control plant, or exhibits increased magnitude of response thanthe control plant when subjected to the same concentration of ABA as thecontrol plant.

“Hyposensitivity” or “decreased response” of a plant to ABA means thatthe plant exhibits ABA induced phenotype at higher concentration of ABAthan the control plant, or exhibits decreased magnitude of response thanthe control plant when subjected to the same concentration of ABA as thecontrol plant.

Sensitivity to ABA can be assessed at various plant developmentalstages. Examples include, but are not limited to, germination, cotyledonexpansion, green cotyledons, expansion of the first true leaf, alteredroot growth rate or developmental arrest in the seedling stage.Moreover, the concentration of ABA at which sensitivity is observedvaries in a species dependent manner. For example, transgenicArabidopsis thaliana will demonstrate sensitivity at a lowerconcentration than observed in Brassica or soybean.

The term “percentage greenness” or “% greenness” refers herein to thepercentage of seedlings that have totally green leaves, wherein thepercentage is calculated with respect to the total number of seedlingsbeing tested. “Percentage greenness” as referred to herein is scored asthe percentage of seedlings with green leaves compared to seedlings withyellow, brown or purple leaves. “Percentage greenness” can be scored at1-leaf or 2-leaf stage for seedlings of a monocot plant, wherein thefirst and second leaves are true leaves. “Percentage greenness” as usedherein, can be scored at 3- or 4-leaf stage for seedlings of a dicotplant, wherein two of the leaves are cotyledonary leaves, and the thirdand fourth leaves are true leaves. To calculate % greenness in theseedlings of a dicot plant, any seedling with any yellow or brownstreaks on any of the four leaves is not considered green. To calculate% greenness in the seedlings of a monocot plant, any seedling with anyyellow or brown streaks on any of the first or second leaves is notconsidered green. In one embodiment of the current disclosure,“percentage greenness” is calculated when all the seedlings aresubjected to osmotic stress.

“True leaves” as used herein refer to the non-cotyledonary leaves of theplant or the seedling.

The term “percentage leaf emergence” or “% leaf emergence” refers hereinto the percentage of seedlings that had fully expanded 1-, 2- or 3-trueleaves, wherein the percentage is calculated with respect to the totalnumber of seedlings being tested. “Percentage leaf emergence” can bescored as the appearance of fully expanded first two true leaves for theseedlings of a dicot plant. “Percentage leaf emergence” can be scored asthe appearance of fully expanded first 1- or 2-true leaves for theseedlings of a monocot plant. In one embodiment of the currentdisclosure, the “percentage leaf emergence” is calculated when all theseedlings are subjected to osmotic stress.

One of ordinary skill in the art is familiar with protocols forsimulating drought conditions and for evaluating drought tolerance ofplants that have been subjected to simulated or naturally-occurringdrought conditions. For example, one can simulate drought conditions bygiving plants less water than normally required or no water over aperiod of time, and one can evaluate drought tolerance by looking fordifferences in physiological and/or physical condition, including (butnot limited to) vigor, growth, size, or root length, or in particular,leaf color or leaf area size. Other techniques for evaluating droughttolerance include measuring chlorophyll fluorescence, photosyntheticrates and gas exchange rates.

A drought stress experiment may involve a chronic stress (i.e., slow drydown) and/or may involve two acute stresses (i.e., abrupt removal ofwater) separated by a day or two of recovery. Chronic stress may last8-10 days. Acute stress may last 3-5 days. The following variables maybe measured during drought stress and well watered treatments oftransgenic plants and relevant control plants:

The variable “% area chg_start chronic-acute2” is a measure of thepercent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and the day of thesecond acute stress.

The variable “% area chg_start chronic-end chronic” is a measure of thepercent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and the last day ofchronic stress.

The variable “% area chg_start chronic-harvest” is a measure of thepercent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and the day of harvest.

The variable “% area chg_start chronic-recovery24 hr” is a measure ofthe percent change in total area determined by remote visible spectrumimaging between the first day of chronic stress and 24 hrs into therecovery (24 hrs after acute stress 2).

The variable “psii_acute1” is a measure of Photosystem II (PSII)efficiency at the end of the first acute stress period. It provides anestimate of the efficiency at which light is absorbed by PSII antennaeand is directly related to carbon dioxide assimilation within the leaf.

The variable “psii_acute2” is a measure of Photosystem II (PSII)efficiency at the end of the second acute stress period. It provides anestimate of the efficiency at which light is absorbed by PSII antennaeand is directly related to carbon dioxide assimilation within the leaf.

The variable “fv/fm_acute1” is a measure of the optimum quantum yield(Fv/Fm) at the end of the first acute stress−(variable fluorescencedifference between the maximum and minimum fluorescence/maximumfluorescence)

The variable “fv/fm_acute2” is a measure of the optimum quantum yield(Fv/Fm) at the end of the second acute stress−(variable fluorescencedifference between the maximum and minimum fluorescence/maximumfluorescence).

The variable “leaf rolling_harvest” is a measure of the ratio of topimage to side image on the day of harvest.

The variable “leaf rolling_recovery24 hr” is a measure of the ratio oftop image to side image 24 hours into the recovery.

The variable “Specific Growth Rate (SGR)” represents the change in totalplant surface area (as measured by Lemna Tec Instrument) over a singleday (Y(t)=Y0*e^(r*t)). Y(t)=Y0*e^(r*t) is equivalent to % change in Y/Δtwhere the individual terms are as follows: Y(t)=Total surface area at t;Y0=Initial total surface area (estimated); r=Specific Growth Rate day⁻¹,and t=Days After Planting (“DAP”).

The variable “shoot dry weight” is a measure of the shoot weight 96hours after being placed into a 104° C. oven.

The variable “shoot fresh weight” is a measure of the shoot weightimmediately after being cut from the plant.

The Examples below describe some representative protocols and techniquesfor simulating drought conditions and/or evaluating drought tolerance.

One can also evaluate drought tolerance by the ability of a plant tomaintain sufficient yield (at least 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% yield) in field testing under simulated ornaturally-occurring drought conditions (e.g., by measuring forsubstantially equivalent yield under drought conditions compared tonon-drought conditions, or by measuring for less yield loss underdrought conditions compared to a control or reference plant).

One of ordinary skill in the art would readily recognize a suitablecontrol or reference plant to be utilized when assessing or measuring anagronomic characteristic or phenotype of a transgenic plant in anyembodiment of the present disclosure in which a control plant isutilized (e.g., compositions or methods as described herein). Forexample, by way of non-limiting illustrations:

1. Progeny of a transformed plant which is hemizygous with respect to arecombinant DNA construct (or suppression DNA construct), such that theprogeny are segregating into plants either comprising or not comprisingthe recombinant DNA construct (or suppression DNA construct): theprogeny comprising the recombinant DNA construct (or suppression DNAconstruct) would be typically measured relative to the progeny notcomprising the recombinant DNA construct (or suppression DNA construct)(i.e., the progeny not comprising the recombinant DNA construct (or thesuppression DNA construct) is the control or reference plant).

2. Introgression of a recombinant DNA construct (or suppression DNAconstruct) into an inbred line, such as in maize, or into a variety,such as in soybean: the introgressed line would typically be measuredrelative to the parent inbred or variety line (i.e., the parent inbredor variety line is the control or reference plant).

3. Two hybrid lines, where the first hybrid line is produced from twoparent inbred lines, and the second hybrid line is produced from thesame two parent inbred lines except that one of the parent inbred linescontains a recombinant DNA construct (or suppression DNA construct): thesecond hybrid line would typically be measured relative to the firsthybrid line (i.e., the first hybrid line is the control or referenceplant).

4. A plant comprising a recombinant DNA construct (or suppression DNAconstruct): the plant may be assessed or measured relative to a controlplant not comprising the recombinant DNA construct (or suppression DNAconstruct) but otherwise having a comparable genetic background to theplant (e.g., sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity of nuclear genetic material comparedto the plant comprising the recombinant DNA construct (or suppressionDNA construct)). There are many laboratory-based techniques availablefor the analysis, comparison and characterization of plant geneticbackgrounds; among these are Isozyme Electrophoresis, RestrictionFragment Length Polymorphisms (RFLPs), Randomly Amplified PolymorphicDNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Amplified Fragment Length Polymorphisms (AFLP®s), andSimple Sequence Repeats (SSRs) which are also referred to asMicrosatellites.

Furthermore, one of ordinary skill in the art would readily recognizethat a suitable control or reference plant to be utilized when assessingor measuring an agronomic characteristic or phenotype of a transgenicplant would not include a plant that had been previously selected, viamutagenesis or transformation, for the desired agronomic characteristicor phenotype.

Methods:

Methods include but are not limited to methods for increasing droughttolerance in a plant, methods for increasing triple stress tolerance ina plant, methods for increasing osmotic stress tolerance in a plant,methods for increasing nitrogen stress tolerance in a plant, methods forevaluating drought tolerance in a plant, methods for evaluating triplestress tolerance in a plant, methods for evaluating osmotic stresstolerance in a plant, methods for evaluating nitrogen stress tolerancein a plant, methods for altering ABA response in a plant, methods forincreasing tiller number in a plant, methods for alteration of rootarchitecture in a plant, methods for evaluating altered ABA response ina plant, methods for altering an agronomic characteristic in a plant,methods for determining an alteration of an agronomic characteristic ina plant, and methods for producing seed. The plant may be amonocotyledonous or dicotyledonous plant, for example, a maize orsoybean plant. The plant may also be sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, millet, sugar cane or sorghum. The seedmay be a maize or soybean seed, for example, a maize hybrid seed ormaize inbred seed.

Methods include but are not limited to the following:

A method for transforming a cell (or microorganism) comprisingtransforming a cell (or microorganism) with any of the isolatedpolynucleotides or recombinant DNA constructs of the present disclosure.The cell (or microorganism) transformed by this method is also included.In particular embodiments, the cell is eukaryotic cell, e.g., a yeast,insect or plant cell, or prokaryotic, e.g., a bacterial cell. Themicroorganism may be Agrobacterium, e.g. Agrobacterium tumefaciens orAgrobacterium rhizogenes.

A method for producing a transgenic plant comprising transforming aplant cell with any of the isolated polynucleotides or recombinant DNAconstructs (including suppression DNA constructs) of the presentdisclosure and regenerating a transgenic plant from the transformedplant cell. The disclosure is also directed to the transgenic plantproduced by this method, and transgenic seed obtained from thistransgenic plant. The transgenic plant obtained by this method may beused in other methods of the present disclosure.

A method for isolating a polypeptide of the disclosure from a cell orculture medium of the cell, wherein the cell comprises a recombinant DNAconstruct comprising a polynucleotide of the disclosure operably linkedto at least one heterologous regulatory sequence, and wherein thetransformed host cell is grown under conditions that are suitable forexpression of the recombinant DNA construct.

A method of altering the level of expression of a polypeptide of thedisclosure in a host cell comprising: (a) transforming a host cell witha recombinant DNA construct of the present disclosure; and (b) growingthe transformed host cell under conditions that are suitable forexpression of the recombinant DNA construct wherein expression of therecombinant DNA construct results in production of altered levels of thepolypeptide of the disclosure in the transformed host cell.

A method of increasing stress tolerance in a plant, wherein the stressis selected from the group consisting of drought stress, triple stress,nitrogen stress and osmotic stress, the method comprising: (a)introducing into a regenerable plant cell a recombinant DNA constructcomprising a polynucleotide operably linked to at least one regulatorysequence (for example, a promoter functional in a plant), wherein thepolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V or Clustal W method of alignment, when comparedto SEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61, 64, 65, 66, 95, 97,101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132,135, 627 or 628; and (b) regenerating a transgenic plant from theregenerable plant cell after step (a), wherein the transgenic plantcomprises in its genome the recombinant DNA construct and exhibitsincreased stress tolerance, wherein the stress is selected from thegroup consisting of drought stress, triple stress, nitrogen stress andosmotic stress, when compared to a control plant not comprising therecombinant DNA construct. The method may further comprise (c) obtaininga progeny plant derived from the transgenic plant, wherein said progenyplant comprises in its genome the recombinant DNA construct and exhibitsincreased stress tolerance, wherein the stress is selected from thegroup consisting of drought stress, triple stress, nitrogen stress andosmotic stress, when compared to a control plant not comprising therecombinant DNA construct.

A method of increasing stress tolerance, wherein the stress is selectedfrom the group consisting of drought stress, triple stress and osmoticstress the method comprising: (a) introducing into a regenerable plantcell a recombinant DNA construct comprising a polynucleotide operablylinked to at least one heterologous regulatory element, wherein saidpolynucleotide comprises a nucleotide sequence, wherein the nucleotidesequence is: (a) hybridizable under stringent conditions with a DNAmolecule comprising the full complement of SEQ ID NO:16, 17, 19, 38, 42,44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112,116, 118, 120 or 122; or (b) derived from SEQ ID NO:16, 17, 19, 38, 42,44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112,116, 118, 120 or 122, by alteration of one or more nucleotides by atleast one method selected from the group consisting of: deletion,substitution, addition and insertion; and (b) regenerating a transgenicplant from the regenerable plant cell after step (a), wherein thetransgenic plant comprises in its genome the recombinant DNA constructand exhibits increased stress tolerance, wherein the stress is selectedfrom the group consisting of drought stress, triple stress, nitrogenstress and osmotic stress, when compared to a control plant notcomprising the recombinant DNA construct. The method may furthercomprise (c) obtaining a progeny plant derived from the transgenicplant, wherein said progeny plant comprises in its genome therecombinant DNA construct and exhibits increased stress tolerance,wherein the stress is selected from the group consisting of droughtstress, triple stress, nitrogen stress and osmotic stress, when comparedto a control plant not comprising the recombinant DNA construct.

A method of selecting for (or identifying) increased stress tolerance ina plant, wherein the stress is selected from the group consisting ofdrought stress, triple stress, nitrogen stress and osmotic stress, themethod comprising (a) obtaining a transgenic plant, wherein thetransgenic plant comprises in its genome a recombinant DNA constructcomprising a polynucleotide operably linked to at least one heterologousregulatory sequence (for example, a promoter functional in a plant),wherein said polynucleotide encodes a polypeptide having an amino acidsequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity, based on the Clustal V or Clustal W method ofalignment, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59,61, 64, 65, 66, 95, 97, 101, 103, 107, 111,113, 117, 119, 121, 123, 127,129, 130, 131, 132, 135, 627 or 628; (b) obtaining a progeny plantderived from said transgenic plant, wherein the progeny plant comprisesin its genome the recombinant DNA construct; and (c) selecting (oridentifying) the progeny plant with increased stress tolerance, whereinthe stress is selected from the group consisting of drought stress,triple stress, nitrogen stress and osmotic stress tolerance, compared toa control plant not comprising the recombinant DNA construct.

In another embodiment, a method of selecting for (or identifying)increased stress tolerance in a plant, wherein the stress is selectedfrom the group consisting of drought stress, triple stress, nitrogenstress and osmotic stress, the method comprising: (a) obtaining atransgenic plant, wherein the transgenic plant comprises in its genome arecombinant DNA construct comprising a polynucleotide operably linked toat least one heterologous regulatory element, wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V or Clustal W method of alignment, when comparedto SEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64, 65, 66, 95, 97,101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132,135, 627 or 628; (b) growing the transgenic plant of part (a) underconditions wherein the polynucleotide is expressed; and (c) selecting(or identifying) the transgenic plant of part (b) with increased stresstolerance, wherein the stress is selected from the group consisting ofdrought stress, triple stress, nitrogen stress and osmotic stress,compared to a control plant not comprising the recombinant DNAconstruct.

A method of selecting for (or identifying) increased stress tolerance ina plant, wherein the stress is selected from the group consisting ofdrought stress, triple stress, nitrogen stress and osmotic stress themethod comprising: (a) obtaining a transgenic plant, wherein thetransgenic plant comprises in its genome a recombinant DNA constructcomprising a polynucleotide operably linked to at least one heterologousregulatory element, wherein said polynucleotide comprises a nucleotidesequence, wherein the nucleotide sequence is: (i) hybridizable understringent conditions with a DNA molecule comprising the full complementof SEQ ID NO:16, 17, 19, 38, 42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94,96, 100, 102, 106, 110, 112, 116, 118, 120 or 122; or (ii) derived fromSEQ ID NO:16, 17, 19, 38, 42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94,96, 100, 102, 106, 110, 112, 116, 118, 120 or 122 by alteration of oneor more nucleotides by at least one method selected from the groupconsisting of: deletion, substitution, addition and insertion; (b)obtaining a progeny plant derived from said transgenic plant, whereinthe progeny plant comprises in its genome the recombinant DNA construct;and (c) selecting (or identifying) the progeny plant with increasedstress tolerance, when compared to a control plant not comprising therecombinant DNA construct.

A method of making a plant that exhibits at least one phenotype selectedfrom the group consisting of: increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number, increased yield and increasedbiomass, when compared to a control plant, the method comprising thesteps of introducing into a plant a recombinant DNA construct comprisinga polynucleotide operably linked to at least one heterologous regulatoryelement, wherein said polynucleotide encodes a polypeptide having anamino acid sequence of at least 80% sequence identity, when compared toSEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64, 65, 66, 95, 97, 101,103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135,627 or 628.

A method of producing a plant that exhibits at least one phenotypeselected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, increased yield andincreased biomass, wherein the method comprises growing a plant from aseed comprising a recombinant DNA construct, wherein the recombinant DNAconstruct comprises a polynucleotide operably linked to at least oneheterologous regulatory element, wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 80% sequenceidentity, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59,61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127,129, 130, 131, 132, 135, 627 or 628, wherein the plant exhibits at leastone phenotype selected from the group consisting of: increased triplestress tolerance, increased drought stress tolerance, increased nitrogenstress tolerance, increased osmotic stress tolerance, altered ABAresponse, altered root architecture, increased tiller number, increasedyield and increased biomass, when compared to a control plant notcomprising the recombinant DNA construct.

A method of making a plant that exhibits at least one phenotype selectedfrom the group consisting of: increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number, increased yield and increasedbiomass, the method comprising: (a) introducing into a regenerable plantcell a recombinant DNA construct comprising a polynucleotide operablylinked to at least one regulatory sequence, wherein the polynucleotideencodes a polypeptide gives an E-value score of 1E-15 or less whenqueried using a Profile Hidden Markov Model prepared using SEQ IDNOS:18, 29, 33, 45, 47, 53, 55, 61, 64, 65, 77, 78, 101, 103, 105, 107,111, 115, 131, 132, 135, 137, 139, 141, 144, 433, 559 and 604, the querybeing carried out using the hmmsearch algorithm wherein the Z parameteris set to 1 billion; (b) regenerating a transgenic plant from theregenerable plant cell of (a), wherein the transgenic plant comprises inits genome the recombinant DNA construct; and (c) obtaining a progenyplant derived from the transgenic plant of (b), wherein said progenyplant comprises in its genome the recombinant DNA construct and exhibitsat least one phenotype selected from the group consisting of: increasedtriple stress tolerance, increased drought stress tolerance, increasednitrogen stress tolerance, increased osmotic stress tolerance, alteredABA response, altered root architecture, increased tiller number,increased yield and increased biomass, when compared to a control plantnot comprising the recombinant DNA construct.

A method of increasing in a crop plant at least one phenotype selectedfrom the group consisting of: triple stress tolerance, drought stresstolerance, nitrogen stress tolerance, osmotic stress tolerance, ABAresponse, tiller number, yield and biomass, the method comprisingincreasing the expression of a carboxyl esterase in the crop plant. Inone embodiment, the crop plant is maize. In one embodiment, thecarboxylesterase has at least 80% sequence identity, when compared toSEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64, 65, 66, 95, 97, 101,103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135,627 or 628. In one embodiment, the carboxylesterase is a DTP4polypeptide disclosed in Table 1 and Table 2 in the current disclosure.In one embodiment, the carboxylesterase gives an E-value score of 1E-15or less when queried using a Profile Hidden Markov Model prepared usingSEQ ID NOS:18, 29, 33, 45, 47, 53, 55, 61, 64, 65, 77, 78, 101, 103,105, 107, 111, 115, 131, 132, 135, 137, 139, 141, 144, 433, 559 and 604,the query being carried out using the hmmsearch algorithm wherein the Zparameter is set to 1 billion.

In one embodiment, the carboxylesterase is a polypeptide wherein thepolypeptide gives an E-value score of 1E-15 or less when queried usingthe Profile Hidden Markov Model given in Table 18.

One embodiment encompasses a method of increasing stress tolerance in aplant, wherein the stress is selected from a group consisting of:drought stress, triple stress, nitrogen stress and osmotic stress, themethod comprising:

(a) introducing into a regenerable plant cell a recombinant DNAconstruct comprising a polynucleotide operably linked to at least oneregulatory sequence, wherein the polynucleotide encodes a polypeptidegives an E-value score of 1E-15 or less when queried using a ProfileHidden Markov Model prepared using SEQ ID NOS:18, 29, 33, 45, 47, 53,55, 61,64, 65, 77, 78, 101, 103, 105, 107, 111, 115, 131,132, 135, 137,139, 141, 144, 433, 559 and 604, the query being carried out using thehmmsearch algorithm wherein the Z parameter is set to 1 billion; (b)regenerating a transgenic plant from the regenerable plant cell of (a),wherein the transgenic plant comprises in its genome the recombinant DNAconstruct; and (c) obtaining a progeny plant derived from the transgenicplant of (b), wherein said progeny plant comprises in its genome therecombinant DNA construct and exhibits increased tolerance to at leastone stress selected from the group consisting of: drought stress, triplestress, nitrogen stress and osmotic stress, when compared to a controlplant not comprising the recombinant DNA construct.

A method of selecting for (or identifying) an alteration of an agronomiccharacteristic in a plant, comprising (a) obtaining a transgenic plant,wherein the transgenic plant comprises in its genome a recombinant DNAconstruct comprising a polynucleotide operably linked to at least oneheterologous regulatory sequence (for example, a promoter functional ina plant), wherein said polynucleotide encodes a polypeptide having anamino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, based on the Clustal V or Clustal W method ofalignment, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55,59, 61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123,127, 129, 130, 131, 132, 135, 627 or 628; (b) obtaining a progeny plantderived from said transgenic plant, wherein the progeny plant comprisesin its genome the recombinant DNA construct; and (c) selecting (oridentifying) the progeny plant that exhibits an alteration in at leastone agronomic characteristic when compared, optionally under at leastone stress condition, to a control plant not comprising the recombinantDNA construct. The at least one stress condition may be selected fromthe group of drought stress, triple stress, nitrogen stress and osmoticstress. The polynucleotide preferably encodes a DTP4 polypeptide. TheDTP4 polypeptide preferably has stress tolerance activity, wherein thestress is selected from the group consisting of drought stress, triplestress, nitrogen stress and osmotic stress.

In another embodiment, a method of selecting for (or identifying) analteration of at least one agronomic characteristic in a plant,comprising: (a) obtaining a transgenic plant, wherein the transgenicplant comprises in its genome a recombinant DNA construct comprising apolynucleotide operably linked to at least one heterologous regulatoryelement, wherein said polynucleotide encodes a polypeptide having anamino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, based on the Clustal V or Clustal W method ofalignment, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55,59, 61, 64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123,127, 129, 130, 131, 132, 135, 627 or 628, wherein the transgenic plantcomprises in its genome the recombinant DNA construct; (b) growing thetransgenic plant of part (a) under conditions wherein the polynucleotideis expressed; and (c) selecting (or identifying) the transgenic plant ofpart (b) that exhibits an alteration of at least one agronomiccharacteristic when compared to a control plant not comprising therecombinant DNA construct. Optionally, said selecting (or identifying)step (c) comprises determining whether the transgenic plant exhibits analteration of at least one agronomic characteristic when compared, underat least one condition, to a control plant not comprising therecombinant DNA construct. The at least one agronomic trait may beyield, biomass, or both and the alteration may be an increase. The atleast one stress condition may be selected from the group of droughtstress, triple stress, nitrogen stress and osmotic stress.

The at least one agronomic characteristic may be abiotic stresstolerance, greenness, yield, growth rate, biomass, fresh weight atmaturation, dry weight at maturation, fruit yield, seed yield, totalplant nitrogen content, fruit nitrogen content, seed nitrogen content,nitrogen content in a vegetative tissue, total plant free amino acidcontent, fruit free amino acid content, seed free amino acid content,free amino acid content in a vegetative tissue, total plant proteincontent, fruit protein content, seed protein content, protein content ina vegetative tissue, drought tolerance, nitrogen uptake, root lodging,harvest index, stalk lodging, plant height, ear height, ear length, leafnumber, tiller number, growth rate, first pollen shed time, first silkemergence time, anthesis silking interval (ASI), stalk diameter, rootarchitecture, staygreen, relative water content, water use, water useefficiency, dry weight of either main plant, tillers, primary ear, mainplant and tillers or cobs; rows of kernels, total plant weight·kernelweight, kernel number, salt tolerance, chlorophyll content, flavonolcontent, number of yellow leaves, early seedling vigor and seedlingemergence under low temperature stress. These agronomic characteristicsmaybe measured at any stage of the plant development. One or more ofthese agronomic characteristics may be measured under stress ornon-stress conditions, and may show alteration on overexpression of therecombinant constructs disclosed herein.

A method of selecting for (or identifying) an alteration of an agronomiccharacteristic in a plant, comprising (a) obtaining a transgenic plant,wherein the transgenic plant comprises in its genome a recombinant DNAconstruct comprising a polynucleotide operably linked to at least oneheterologous regulatory element, wherein said polynucleotide comprises anucleotide sequence, wherein the nucleotide sequence is: (i)hybridizable under stringent conditions with a DNA molecule comprisingthe full complement of SEQ ID NO:16, 17, 19, 38, 42, 44, 46, 48, 50, 54,58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112, 116, 118, 120 or 122;or (ii) derived from SEQ ID NO:16, 17, 19, 38, 42, 44, 46, 48, 50, 54,58, 60, 62, 63, 94, 96, 100, 102, 106, 110, 112, 116, 118, 120 or 122 byalteration of one or more nucleotides by at least one method selectedfrom the group consisting of: deletion, substitution, addition andinsertion; (b) obtaining a progeny plant derived from said transgenicplant, wherein the progeny plant comprises in its genome the recombinantDNA construct; and (c) selecting (or identifying) the progeny plant thatexhibits an alteration in at least one agronomic characteristic whencompared, optionally under stress conditions, wherein the stress isselected from the group consisting of drought stress, triple stress,nitrogen stress and osmotic stress, to a control plant not comprisingthe recombinant DNA construct. The polynucleotide preferably encodes aDTP4 polypeptide. The DTP4 polypeptide preferably has stress toleranceactivity, wherein the stress is selected from the group consisting ofdrought stress, triple stress, nitrogen stress and osmotic stress.

The use of a recombinant DNA construct for producing a plant thatexhibits at least one phenotype selected from the group consisting of:increased triple stress tolerance, increased drought stress tolerance,increased nitrogen stress tolerance, increased osmotic stress tolerance,altered ABA response, altered root architecture, increased tillernumber, increased yield and increased biomass, when compared to acontrol plant not comprising said recombinant DNA construct, wherein therecombinant DNA construct comprises a polynucleotide operably linked toat least one heterologous regulatory element, wherein the polynucleotideencodes a polypeptide having an amino acid sequence of at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity, based on the Clustal V or the Clustal W method ofalignment, using the respective default parameters, when compared to SEQID NO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101,103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135,627 or 628. The polypeptide may be over-expressed in at least one tissueof the plant, or during at least one condition of environmental stress,or both. The plant may be selected from the group consisting of: maize,soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,barley, millet, sugar cane and switchgrass.

A method of producing seed (for example, seed that can be sold as adrought tolerant product offering) comprising any of the precedingmethods, and further comprising obtaining seeds from said progeny plant,wherein said seeds comprise in their genome said recombinant DNAconstruct (or suppression DNA construct).

A method of producing oil or a seed by-product, or both, from a seed,the method comprising extracting oil or a seed by-product, or both, froma seed that comprises a recombinant DNA construct, wherein therecombinant DNA construct comprises a polynucleotide operably linked toat least one heterologous regulatory element, wherein the polynucleotideencodes a polypeptide having an amino acid sequence of at least 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity, based on the Clustal V or the Clustal W method ofalignment, using the respective default parameters, when compared to SEQID NO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61,64, 65, 66, 95, 97, 101,103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135,627 or 628. The seed may be obtained from a plant that comprises therecombinant DNA construct, wherein the plant exhibits at least onephenotype selected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, increased yield andincreased biomass, when compared to a control plant not comprising therecombinant DNA construct. The polypeptide may be over-expressed in atleast one tissue of the plant, or during at least one condition ofabiotic stress, or both. The plant may be selected from the groupconsisting of: maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass. Theoil or the seed by-product, or both, may comprise the recombinant DNAconstruct.

Methods of isolating seed oils are well known in the art: (Young et al.,Processing of Fats and Oils, In The Lipid Handbook, Gunstone et al.,eds., Chapter 5 pp 253 257; Chapman & Hall: London (1994)). Seedby-products include but are not limited to the following: meal,lecithin, gums, free fatty acids, pigments, soap, stearine, tocopherols,sterols and volatiles.

One may evaluate altered root architecture in a controlled environment(e.g., greenhouse) or in field testing. The evaluation may be undersimulated or naturally-occurring low or high nitrogen conditions. Thealtered root architecture may be an increase in root mass. The increasein root mass may be at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%,40%, 45% or 50%, when compared to a control plant not comprising therecombinant DNA construct.

In any of the foregoing methods or any other embodiments of methods ofthe present disclosure, the step of selecting an alteration of anagronomic characteristic in a transgenic plant, if applicable, maycomprise selecting a transgenic plant that exhibits an alteration of atleast one agronomic characteristic when compared, under varyingenvironmental conditions, to a control plant not comprising therecombinant DNA construct.

In any of the foregoing methods or any other embodiments of methods ofthe present disclosure, the step of selecting an alteration of anagronomic characteristic in a progeny plant, if applicable, may compriseselecting a progeny plant that exhibits an alteration of at least oneagronomic characteristic when compared, under varying environmentalconditions, to a control plant not comprising the recombinant DNAconstruct.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, in said introducing step said regenerable plantcell may comprise a callus cell, an embryogenic callus cell, a gameticcell, a meristematic cell, or a cell of an immature embryo. Theregenerable plant cells may derive from an inbred maize plant.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, said regenerating step may comprise thefollowing: (i) culturing said transformed plant cells in a mediacomprising an embryogenic promoting hormone until callus organization isobserved; (ii) transferring said transformed plant cells of step (i) toa first media which includes a tissue organization promoting hormone;and (iii) subculturing said transformed plant cells after step (ii) ontoa second media, to allow for shoot elongation, root development or both.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, the at least one agronomic characteristic may beselected from the group consisting of: abiotic stress tolerance,greenness, yield, growth rate, biomass, fresh weight at maturation, dryweight at maturation, fruit yield, seed yield, total plant nitrogencontent, fruit nitrogen content, seed nitrogen content, nitrogen contentin a vegetative tissue, total plant free amino acid content, fruit freeamino acid content, seed free amino acid content, free amino acidcontent in a vegetative tissue, total plant protein content, fruitprotein content, seed protein content, protein content in a vegetativetissue, drought tolerance, nitrogen uptake, root lodging, harvest index,stalk lodging, plant height, ear height, ear length, leaf number, tillernumber, growth rate, first pollen shed time, first silk emergence time,anthesis silking interval (ASI), stalk diameter, root architecture,staygreen, relative water content, water use, water use efficiency, dryweight of either main plant, tillers, primary ear, main plant andtillers or cobs; rows of kernels, total plant weight·kernel weight,kernel number, salt tolerance, chlorophyll content, flavonol content,number of yellow leaves, early seedling vigor and seedling emergenceunder low temperature stress. The alteration of at least one agronomiccharacteristic may be an increase in yield, greenness or biomass.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, the plant may exhibit the alteration of at leastone agronomic characteristic when compared, under stress conditions,wherein the stress is selected from the group consisting of droughtstress, triple stress, nitrogen stress and osmotic stress, to a controlplant not comprising said recombinant DNA construct (or said suppressionDNA construct).

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, alternatives exist for introducing into aregenerable plant cell a recombinant DNA construct comprising apolynucleotide operably linked to at least one regulatory sequence. Forexample, one may introduce into a regenerable plant cell a regulatorysequence (such as one or more enhancers, optionally as part of atransposable element), and then screen for an event in which theregulatory sequence is operably linked to an endogenous gene encoding apolypeptide of the instant disclosure.

The introduction of recombinant DNA constructs of the present disclosureinto plants may be carried out by any suitable technique, including butnot limited to direct DNA uptake, chemical treatment, electroporation,microinjection, cell fusion, infection, vector-mediated DNA transfer,bombardment, or Agrobacterium-mediated transformation. Techniques forplant transformation and regeneration have been described inInternational Patent Publication WO 2009/006276, the contents of whichare herein incorporated by reference.

The development or regeneration of plants containing the foreign,exogenous isolated nucleic acid fragment that encodes a protein ofinterest is well known in the art. The regenerated plants may beself-pollinated to provide homozygous transgenic plants. Otherwise,pollen obtained from the regenerated plants is crossed to seed-grownplants of agronomically important lines. Conversely, pollen from plantsof these important lines is used to pollinate regenerated plants. Atransgenic plant of the present disclosure containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

EMBODIMENTS

1. A plant comprising in its genome a recombinant DNA constructcomprising a polynucleotide operably linked to at least one heterologousregulatory element, wherein said polynucleotide encodes a polypeptidehaving an amino acid sequence of at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, when compared to SEQ ID NO:18, 39, 43, 45, 47,49, 51, 55, 59, 61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117,119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628, and whereinsaid plant exhibits at least one phenotype selected from the groupconsisting of: increased triple stress tolerance, increased droughtstress tolerance, increased nitrogen stress tolerance, increased osmoticstress tolerance, altered ABA response, altered root architecture, andincreased tiller number, when compared to a control plant not comprisingsaid recombinant DNA construct.

2. A plant comprising in its genome a recombinant DNA constructcomprising a polynucleotide operably linked to at least one heterologousregulatory element, wherein said polynucleotide encodes a polypeptidehaving an amino acid sequence of at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, when compared to SEQ ID NO:18, 39, 43, 45, 47,49, 51, 55, 59, 61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117,119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628, and whereinsaid plant exhibits an increase in yield, biomass, or both, whencompared to a control plant not comprising said recombinant DNAconstruct.

3. The plant of embodiment 2, wherein said plant exhibits said increasein yield, biomass, or both when compared, under water limitingconditions, to said control plant not comprising said recombinant DNAconstruct.

4. The plant of any one of embodiments 1 to 3, wherein said plant isselected from the group consisting of: Arabidopsis, maize, soybean,sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley,millet, sugar cane and switchgrass.

5. Seed of the plant of any one of embodiments 1 to 4, wherein said seedcomprises in its genome a recombinant DNA construct comprising apolynucleotide operably linked to at least one heterologous regulatoryelement, wherein said polynucleotide encodes a polypeptide having anamino acid sequence of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49,51, 55, 59, 61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119,121, 123, 127, 129, 130, 131, 132, 135, 627 or 628, and wherein a plantproduced from said seed exhibits an increase in at least one phenotypeselected from the group consisting of: drought stress tolerance, triplestress tolerance, osmotic stress tolerance, nitrogen stress tolerance,tiller number, yield and biomass, when compared to a control plant notcomprising said recombinant DNA construct.

6. A method of increasing stress tolerance in a plant, wherein thestress is selected from a group consisting of: drought stress, triplestress, nitrogen stress and osmotic stress, the method comprising:

-   -   (a) introducing into a regenerable plant cell a recombinant DNA        construct comprising a polynucleotide operably linked to at        least one heterologous regulatory sequence, wherein the        polynucleotide encodes a polypeptide having an amino acid        sequence of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,        88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or        100% sequence identity, when compared to SEQ ID NO:18, 39, 43,        45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101, 103, 107,        111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627        or 628;    -   (b) regenerating a transgenic plant from the regenerable plant        cell of (a), wherein the transgenic plant comprises in its        genome the recombinant DNA construct; and    -   (c) obtaining a progeny plant derived from the transgenic plant        of (b), wherein said progeny plant comprises in its genome the        recombinant DNA construct and exhibits increased tolerance to at        least one stress selected from the group consisting of drought        stress, triple stress, nitrogen stress and osmotic stress, when        compared to a control plant not comprising the recombinant DNA        construct.

7. A method of selecting for increased stress tolerance in a plant,wherein the stress is selected from a group consisting of: droughtstress, triple stress, nitrogen stress and osmotic stress, the methodcomprising:

-   -   (a) obtaining a transgenic plant, wherein the transgenic plant        comprises in its genome a recombinant DNA construct comprising a        polynucleotide operably linked to at least one heterologous        regulatory element, wherein said polynucleotide encodes a        polypeptide having an amino acid sequence of at least 80%, 81%,        82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,        95%, 96%, 97%, 98%, 99%, or 100% sequence identity, when        compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64,        65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123,        127, 129, 130, 131, 132, 135, 627 or 628;    -   (b) growing the transgenic plant of part (a) under conditions        wherein the polynucleotide is expressed; and    -   (c) selecting the transgenic plant of part (b) with increased        stress tolerance, wherein the stress is selected from the group        consisting of: drought stress, triple stress, nitrogen stress        and osmotic stress, when compared to a control plant not        comprising the recombinant DNA construct.

8. A method of selecting for an alteration of yield, biomass, or both ina plant, comprising:

-   -   (a) obtaining a transgenic plant, wherein the transgenic plant        comprises in its genome a recombinant DNA construct comprising a        polynucleotide operably linked to at least one regulatory        element, wherein said polynucleotide encodes a polypeptide        having an amino acid sequence of at least 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, 99%, or 100% sequence identity, when compared to SEQ        ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64, 65, 66, 95, 97,        101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131,        132, 135, 627 or 628;    -   (b) growing the transgenic plant of part (a) under conditions        wherein the polynucleotide is expressed; and    -   (c) selecting the transgenic plant of part (b) that exhibits an        alteration of yield, biomass or both when compared to a control        plant not comprising the recombinant DNA construct.

9. The method of embodiment 8, wherein said selecting step (c) comprisesdetermining whether the transgenic plant of (b) exhibits an alterationof yield, biomass or both when compared, under water limitingconditions, to a control plant not comprising the recombinant DNAconstruct.

10. The method of embodiment 8 or embodiment 9, wherein said alterationis an increase.

11. The method of any one of embodiments 6 to 10, wherein said plant isselected from the group consisting of: Arabidopsis, maize, soybean,sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley,millet, sugar cane and switchgrass.

12. An isolated polynucleotide comprising:

-   -   (a) a nucleotide sequence encoding a polypeptide with stress        tolerance activity, wherein the stress is selected from a group        consisting of drought stress, triple stress, nitrogen stress and        osmotic stress, and wherein the polypeptide has an amino acid        sequence of at least 95%, 96%, 97%, 98%, 99% or 100% sequence        identity when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51,        55, 59, 61, 64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117,        119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628; or    -   (b) the full complement of the nucleotide sequence of (a).

13. The polynucleotide of embodiment 12, wherein the amino acid sequenceof the polypeptide comprises less than 100% sequence identity to SEQ IDNO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101, 103,107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or628.

14. The polynucleotide of embodiment 12 wherein the nucleotide sequencecomprises SEQ ID NO:16, 17, 19, 38, 42, 44, 46, 48, 50, 54, 58, 60, 62,63, 94, 96, 100, 102, 106, 110, 112, 116, 118, 120 or 122.

15. A plant or seed comprising a recombinant DNA construct, wherein therecombinant DNA construct comprises the polynucleotide of any one ofembodiments 12 to 14 operably linked to at least one heterologousregulatory sequence.

16. A plant comprising in its genome an endogenous polynucleotideoperably linked to at least one heterologous regulatory element, whereinsaid endogenous polynucleotide encodes a polypeptide having an aminoacid sequence of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59,61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127,129, 130, 131, 132, 135, 627 or 628, and wherein said plant exhibits atleast one phenotype selected from the group consisting of increasedtriple stress tolerance, increased drought stress tolerance, increasednitrogen stress tolerance, increased osmotic stress tolerance, alteredABA response, altered root architecture, increased tiller number, whencompared to a control plant not comprising the heterologous regulatoryelement.

17. A method of increasing in a crop plant at least one phenotypeselected from the group consisting of: triple stress tolerance, droughtstress tolerance, nitrogen stress tolerance, osmotic stress tolerance,ABA response, tiller number, yield and biomass, the method comprisingincreasing the expression of a carboxyl esterase in the crop plant.

18. The method of embodiment 17, wherein the crop plant is maize.

19. The method of embodiment 17 or embodiment 18, wherein the carboxylesterase has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59,61, 64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123,127, 129, 130, 131, 132, 135, 627 or 628. The carboxyl esterase maycomprise at least one of the elements present in consensus SEQ ID NO:630selected from the group consisting of: a conserved “nucleophile elbow”(G×S×G), a conserved catalytic triad of S-H-D and a “oxyanion hole” withthe conserved residues G-G-G.

20. The method of embodiment 17 or embodiment 18, wherein thecarboxylesterase gives an E-value score of 1E-15 or less when queriedusing a Profile Hidden Markov Model prepared using SEQ ID NOS:18, 29,33, 45, 47, 53, 55, 61, 64, 65, 77, 78, 101, 103, 105, 107, 111, 115,131, 132, 135, 137, 139, 141, 144, 433, 559 and 604, the query beingcarried out using the hmmsearch algorithm wherein the Z parameter is setto 1 billion.

21. A recombinant DNA construct comprising a polynucleotide, wherein thepolynucleotide is operably linked to a heterologous promoter, andencodes a polypeptide with at least one activity selected from the groupconsisting of: carboxylesterase, increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number, wherein the polypeptide gives anE-value score of 1E-15 or less when queried using a Profile HiddenMarkov Model prepared using SEQ ID NOS:18, 29, 33, 45, 47, 53, 55, 61,64, 65, 77, 78, 101, 103, 105, 107, 111, 115, 131, 132, 135, 137, 139,141, 144, 433, 559 and 604, the query being carried out using thehmmsearch algorithm wherein the Z parameter is set to 1 billion.

22. A plant comprising the recombinant construct of embodiment 21,wherein the plant exhibits increased yield, biomass, or both, whencompared to a plant not comprising the recombinant construct.

23. A method of making a plant, that exhibits at least one phenotypeselected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, the methodcomprising:

-   -   (a) introducing into a regenerable plant cell the recombinant        DNA construct of embodiment 21;    -   (b) regenerating a transgenic plant from the regenerable plant        cell of (a), wherein the transgenic plant comprises in its        genome the recombinant DNA construct; and    -   (c) obtaining a progeny plant derived from the transgenic plant        of (b), wherein said progeny plant comprises in its genome the        recombinant DNA construct of embodiment 21 and exhibits at least        one phenotype selected from the group consisting of: increased        triple stress tolerance, increased drought stress tolerance,        increased nitrogen stress tolerance, increased osmotic stress        tolerance, altered ABA response, altered root architecture,        increased tiller number, when compared to a control plant not        comprising the recombinant DNA construct.

24. A method of increasing stress tolerance in a plant, wherein thestress is selected from a group consisting of: drought stress, triplestress, nitrogen stress and osmotic stress, the method comprising:

-   -   (a) introducing into a regenerable plant cell the recombinant        DNA construct of embodiment 21;    -   (b) regenerating a transgenic plant from the regenerable plant        cell of (a), wherein the transgenic plant comprises in its        genome the recombinant DNA construct; and    -   (c) obtaining a progeny plant derived from the transgenic plant        of (b), wherein said progeny plant comprises in its genome the        recombinant DNA construct of embodiment 21 and exhibits        increased tolerance to at least one stress selected from the        group consisting of: drought stress, triple stress, nitrogen        stress and osmotic stress, when compared to a control plant not        comprising the recombinant DNA construct.

25. A method of making a plant that exhibits at least one phenotypeselected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, increased yield andincreased biomass, when compared to a control plant, the methodcomprising the steps of introducing into a plant a recombinant DNAconstruct comprising a polynucleotide operably linked to at least oneheterologous regulatory element, wherein said polynucleotide encodes apolypeptide having an amino acid sequence of at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity, when compared to SEQ ID NO:18,39, 43, 45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101, 103, 107,111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628.

26. A method of producing a plant that exhibits at least one traitselected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, increased yield andincreased biomass, wherein the method comprises growing a plant from aseed comprising a recombinant DNA construct, wherein the recombinant DNAconstruct comprises a polynucleotide operably linked to at least oneheterologous regulatory element, wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity, when compared to SEQ ID NO:18,39, 43, 45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101, 103, 107,111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or 628,wherein the plant exhibits at least one phenotype selected from thegroup consisting of: increased triple stress tolerance, increaseddrought stress tolerance, increased nitrogen stress tolerance, increasedosmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number, increased yield and increasedbiomass, when compared to a control plant not comprising the recombinantDNA construct.

27. A method of producing a seed, the method comprising the following:

-   -   (a) crossing a first plant with a second plant, wherein at least        one of the first plant and the second plant comprises a        recombinant DNA construct, wherein the recombinant DNA construct        comprises a polynucleotide operably linked to at least one        heterologous regulatory element, wherein the polynucleotide        encodes a polypeptide having an amino acid sequence of at least        80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,        when compared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59,        61,64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121,        123, 127, 129, 130, 131, 132, 135, 627 or 628; and    -   (b) selecting a seed of the crossing of step (a), wherein the        seed comprises the recombinant DNA construct.

28. The method of embodiment 27, wherein a plant grown from the seed ofpart (b) exhibits at least one phenotype selected from the groupconsisting of: increased triple stress tolerance, increased droughtstress tolerance, increased nitrogen stress tolerance, increased osmoticstress tolerance, altered ABA response, altered root architecture,increased tiller number, increased yield and increased biomass, whencompared to a control plant not comprising the recombinant DNAconstruct.

29. A method of producing oil or a seed by-product, or both, from aseed, the method comprising extracting oil or a seed by-product, orboth, from a seed that comprises a recombinant DNA construct, whereinthe recombinant DNA construct comprises a polynucleotide operably linkedto at least one heterologous regulatory element, wherein thepolynucleotide encodes a polypeptide having an amino acid sequence of atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, whencompared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61,64, 65, 66,95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131,132, 135, 627 or 628.

30. The method of embodiment 29, wherein the seed is obtained from aplant that comprises the recombinant DNA construct and exhibits at leastone trait selected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, increased yield andincreased biomass, when compared to a control plant not comprising therecombinant DNA construct.

31. The method of embodiment 29 or embodiment 30, wherein the oil or theseed by-product, or both, comprises the recombinant DNA construct.

32. A plant comprising in its genome a recombinant DNA constructcomprising a polynucleotide operably linked to at least one heterologousregulatory element, wherein said polynucleotide encodes a polypeptidehaving an amino acid sequence of at least 95% sequence identity, whencompared to SEQ ID NO:18, and wherein said plant exhibits at least onephenotype selected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, increased yield andincreased biomass, when compared to a control plant not comprising saidrecombinant DNA construct. The amino acid sequence of the polypeptidemay have less than 100% sequence identity to SEQ ID NO:18.

33. A method of making a plant that exhibits at least one phenotypeselected from the group consisting of: increased triple stresstolerance, increased drought stress tolerance, increased nitrogen stresstolerance, increased osmotic stress tolerance, altered ABA response,altered root architecture, increased tiller number, increased yield andincreased biomass, when compared to a control plant, the methodcomprising the steps of introducing into a plant a recombinant DNAconstruct comprising a polynucleotide operably linked to at least oneheterologous regulatory element, wherein said polynucleotide encodes apolypeptide having an amino acid sequence of at least 95% sequenceidentity, when compared to SEQ ID NO:18. The amino acid sequence of thepolypeptide may have less than 100% sequence identity to SEQ ID NO:18.

In any of the above embodiments 1-33, the polypeptide may comprise atleast one of the elements present in consensus SEQ ID NO:630 selectedfrom the group consisting of: a conserved “nucleophile elbow” (G×S×G), aconserved catalytic triad of S-H-D and a “oxyanion hole” with theconserved residues G-G-G.

EXAMPLES

The present disclosure is further illustrated in the following Examples,in which parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating embodiments of the disclosure, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisdisclosure, and without departing from the spirit and scope thereof, canmake various changes and modifications of the disclosure to adapt it tovarious usages and conditions. Thus, various modifications of thedisclosure in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Creation of an Arabidopsis Population with Activation-TaggedGenes

Arabidopsis activation-tagged populations were created using knownmethods. The resulting T1 seed were sown on soil, and transgenicseedlings were selected by spraying with glufosinate (Finale®; AgrEvo;Bayer Environmental Science). A total of 100,000 glufosinate resistantT1 seedlings were selected. T2 seed from each line was kept separate.

Example 2 Screens to Identify Lines with Enhanced Drought Tolerance

Activation-tagged lines can be subjected to a quantitative droughtstress screen (PCT Publication No. WO/2012/058528). Lines with asignificant delay in yellow color accumulation and/or with significantmaintenance of rosette leaf area, when compared to the average of thewhole flat, are designated as Phase 1 hits. Phase 1 hits are re-screenedin duplicate under the same assay conditions. When either or both of thePhase 2 replicates show a significant difference (score of greater than0.9) from the whole flat mean, the line is then considered a validateddrought tolerant line.

Example 3 Screen to Identify Lines with Enhanced ABA Hypersensitivity

The activation tagged lines described in Example 1 can be subjected toindependent ABA sensitivity screens. The screen is done as described inInternational Patent Application No. PCT/US12/62374.

Screening of transgenic plant lines is done on medium supplemented withlow concentration of ABA.

Wild-type and most of transgenic seeds display consistent germinationprofiles with 0.6 μM ABA. Therefore 0.6 μM ABA is used for phase 1mutant screen.

Germination is scored as the emergence of radicle over a period of 3days. Seeds are counted manually using a magnifying lens. The data isanalyzed as percentage germination to the total number of seeds thatwere inoculated. The germination curves are plotted. Like wild-type,most of the transgenic lines have >90% of germination rate at Day 3.Therefore for a line to qualify as outlier, it has to show asignificantly lower germination rate (<75%) at Day 3. Usually the cutoffvalue (75% germination rate) is at least four SD away from the averagevalue of the 96 lines. Data for germination count of all lines and theirgraphs at 48 hrs, 72 hrs is documented.

Example 4 Identification of Activation-Tagged AT-DTP4 Polypeptide Genefrom the Drought Tolerant Activation-Tagged Line

An activation-tagged line (No. 121463) showing drought tolerance wasfurther analyzed. DNA from the line was extracted, and genes flankingthe insert in the mutant line were identified using SAIFF PCR (Siebertet al., Nucleic Acids Res. 23:1087-1088 (1995)). A PCR amplifiedfragment was identified that contained T-DNA border sequence andArabidopsis genomic sequence. Genomic sequence flanking the insert wasobtained, and the candidate gene was identified by alignment to thecompleted Arabidopsis genome. For a given integration event, theannotated gene nearest the 35S enhancer elements/insert was thecandidate for gene that is activated in the line. In the case of line121463, the gene nearest the 35S enhancers at the integration site wasAt5g62180 (SEQ ID NO:16; NCBI GI No. 30697645), encoding a DTP4polypeptide (SEQ ID NO:18; NCBI GI No. 75180635).

Example 5 Identification of Activation-Tagged AT-DTP4 Polypeptide Genefrom the Activation-Tagged Line Showing ABA-Hypersensitivity

An activation-tagged line (No. 990013; 35S0059G11) showingABA-hypersensitivity was further analyzed. DNA from the line wasextracted, and genes flanking the insert in the mutant line wereidentified using SAIFF PCR (Siebert et al., Nucleic Acids Res.23:1087-1088 (1995)). A PCR amplified fragment was identified thatcontained T-DNA border sequence and Arabidopsis genomic sequence.Genomic sequence flanking the insert was obtained, and the candidategene was identified by alignment to the completed Arabidopsis genome.For a given integration event, the annotated gene nearest the 35Senhancer elements/junction was the candidate for gene that is activatedin the line. In the case of line 990013, the gene nearest the 35Senhancers at the integration site was At5g62180 (SEQ ID NO:16; NCBI GINo. 30697645), encoding a DTP4 polypeptide (SEQ ID NO:18; NCBI GI No.75180635).

Example 6 Validation of Arabidopsis Candidate Gene At5g62180 (AT-DTP4Polypeptide) for Drought Tolerance

Candidate genes can be transformed into Arabidopsis and overexpressedunder the 35S promoter (PCT Publication No. WO/2012/058528). If the sameor similar phenotype is observed in the transgenic line as in the parentactivation-tagged line, then the candidate gene is considered to be avalidated “lead gene” in Arabidopsis.

The candidate Arabidopsis DTP4 polypeptide gene (At5g62180; SEQ IDNO:16; NCBI GI No. 30697645) was tested for its ability to conferdrought tolerance.

The candidate gene was cloned behind the 35S promoter in pBC-yellow tocreate the 35S promoter::At5g62180 expression construct,pBC-Yellow-At5g62180.

Transgenic T1 seeds were selected by yellow fluorescence, and T1 seedswere plated next to wild-type seeds and grown under water limitingconditions. Growth conditions and imaging analysis were as described inExample 2. It was found that the original drought tolerance phenotypefrom activation tagging could be recapitulated in wild-type Arabidopsisplants that were transformed with a construct where At5g62180 wasdirectly expressed by the 35S promoter. The drought tolerance score, asdetermined by the method of PCT Publication No. WO/2012/058528, was1.35.

Example 7 Validation of Arabidopsis Candidate Gene At5g62180 (AT-DTP4Polypeptide) for ABA-Hypersensitivity Via Transformation intoArabidopsis

The candidate Arabidopsis DTP4 polypeptide gene (At5g62180; SEQ IDNO:16; NCBI GI No. 30697645) was tested for its ability to conferABA-hypersensitivity in the following manner.

The At5g62180 cDNA protein-coding region was synthesized and cloned intothe transformation vector.

Transgenic T1 seeds were selected, and used for the germination assay asdescribed below. It was found that the original ABA hypersensitivityphenotype could be recapitulated in wild-type Arabidopsis plants thatwere transformed with a construct where At5g62180 was directly expressedby the 35S promoter.

Assay Conditions:

Seeds were surface sterilized and stratified for 96 hrs. About 100 seedswere inoculated in one plate and stratified for 96 hrs, then cultured ina growth chamber programmed for 16 h of light at 22° C. temperature and50% relative humidity. Germination was scored as the emergence ofradicle.

Observations and Results:

Germination was scored as the emergence of radicle in ½ MS media and 1μM ABA over a period of 4 days. Seeds were counted manually using amagnifying lens. The data was analyzed as percentage germination to thetotal number of seeds that were inoculated. The cut-off value was atleast 2 StandDev below control. The germination curves were plotted.Wild-type col-0 plants had >90% of germination rate at Day 3. The linewith pBC-yellow-At5g62180 showed <75% germination on Day 3, as shown inFIG. 4.

Example 8 Characterization of cDNA Clones Encoding DTP4 Polypeptides

cDNA libraries representing mRNAs from various tissues of Zea mays,Dennstaedtia punctilobula, Sesbania bispinosa, Artemisia tridentata,Lamium amplexicaule, Delosperma nubigenum, Peperomia caperata, and otherplant species were prepared and cDNA clones encoding DTP4 polypeptideswere identified.

Table 3 gives additional information about some of the other DTP4polypeptides disclosed herein.

TABLE 3 Description of Some DTP4 Polypeptides SEQ ID NO (aa sequence)Contig Description 119 Bn_Bo assembled contig from Brassica napus andBrassica oleracea ESTs 121 Bole_someBnap_prot assembled contig fromBrassica napus and Brassica oleracea ESTs 123 B-napus_2-1 assembledcontig from more than one Brassica napus ESTs 125 Csinensis plusassembled contig from Citrus sinensis and Citrus clementina 137GSVIVT01027568001; Vitis vinifera 139 GSVIVT01027566001 Vitis vinifera141 GSVIVT01027569001 Vitis vinifera

The BLAST search using the AT-DTP4 polypeptide and maize sequences fromclones listed in Table 1 revealed similarity of the polypeptides encodedby the cDNAs to the DTP4 polypeptides from various organisms. As shownin Table 1, Table 2 and FIG. 1, certain cDNAs encoded polypeptidessimilar to DTP4 polypeptide from Arabidopsis (GI No. 75180635; SEQ IDNO:18). Shown in Table 4 and Table 5 (patent literature) are the BLASTresults for some of the DTP4 polypeptides disclosed herein, that are oneor more of the following: individual Expressed Sequence Tag (“EST”), thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“Full-Insert Sequence” or “FIS”), the sequences of contigsassembled from two or more EST, FIS or PCR sequences (“Contig”), orsequences encoding an entire or functional protein derived from an FISor a contig (“Complete Gene Sequence” or “CGS”). Also shown in Table 4and 5 are the percent sequence identity values for each pair of aminoacid sequences using the Clustal V method of alignment with defaultparameters.

TABLE 4 BLASTP Results for DTP4 polypeptides BLASTP Percent SequenceNCBI GI No. pLog of Sequence (SEQ ID NO) Status (SEQ ID NO) E-valueIdentity cfp2n.pk010.p21 FIS 194704970 >180 100 (SEQ ID NO: 21) (SEQ IDNO: 82) cfp2n.pk070.m7 FIS 195636334 >180 100 (SEQ ID NO: 23) (SEQ IDNO: 84) cfp3n.pk007.i9 FIS 194707422 >180 99.7 (SEQ ID NO: 25) (SEQ IDNO: 86) pco524093 CGS 223948401 >180 100 (SEQ ID NO: 27) (SEQ ID NO: 88)Maize_DTP4-1 CGS 194704970 >180 100 (SEQ ID NO: 29) (SEQ ID NO: 82)Maize_DTP4-2 CGS 23495723 >180 68.2 (SEQ ID NO: 31) (SEQ ID NO: 90)Maize_DTP4-3 CGS 215768720 >180 73.6 (SEQ ID NO: 33) (SEQ ID NO: 92)

TABLE 5 BLASTP Results for DTP4 polypeptides BLASTP Percent SequenceReference pLog of Sequence (SEQ ID NO) Status (SEQ ID NO) E-valueIdentity At5g62180 CGS SEQ ID NO: 12 of >180 100 U.S. Pat. No. 7,915,050(SEQ ID NO: 81) cfp2n.pk010.p21 FIS SEQ ID NO: >180 97.6 (SEQ ID NO: 21)260345 of US20120216318 (SEQ ID NO: 83) cfp2n.pk070.m7 FIS SEQ IDNO: >180 100 (SEQ ID NO: 23) 331675 of US20120216318 (SEQ ID NO: 85)cfp3n.pk007.i9 FIS SEQ ID NO: 7332 >180 97.6 (SEQ ID NO: 25) of U.S.Pat. No. 8,343,764 (SEQ ID NO: 87) pco524093 CGS SEQ ID NO: 16159 >180100 (SEQ ID NO: 27) of U.S. Pat. No. 7,569,389 (SEQ ID NO: 89)Maize_DTP4-1 CGS SEQ ID NO: >180 97.6 (SEQ ID NO: 29) 260345 ofUS20120216318 (SEQ ID NO: 83) Maize_DTP4-2 CGS SEQ ID NO: 50819 >180 100(SEQ ID NO: 31) of US20120017292 (SEQ ID NO: 91) Maize_DTP4-3 CGS SEQ IDNO: 10044 >180 90 (SEQ ID NO: 33) of U.S. Pat. No. 8,362,325 (SEQ ID NO:93)

FIG. 1A-FIG. 1G show the alignment of the DTP4 polypeptides which weretested in ABA sensitivity assays (SEQ ID NOS:18, 39, 43, 45, 47, 49, 51,55, 59, 61, 64, 65, 66, 95, 97, 99, 101, 103, 107, 111, 113, 117, 119,121,123, 127, 129, 130, 131, 132, 135, 627 and 628). Residues that areidentical to the residue of consensus sequence (SEQ ID NO:630) at agiven position are enclosed in a box. A consensus sequence is presentedwhere a residue is shown if identical in all sequences, otherwise, aperiod is shown.

FIG. 2 shows the percent sequence identity and the divergence values foreach pair of amino acids sequences of DTP4 polypeptides displayed inFIG. 1A-1G.

Sequence alignments and percent identity calculations were performedusing the Megalign® program of the LASERGENE® bioinformatics computingsuite (DNASTAR® Inc., Madison, Wis.). Multiple alignment of thesequences was performed using the Clustal V method of alignment (Higginsand Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method were KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

Sequence alignments and BLAST scores and probabilities indicate that thenucleic acid fragments comprising the instant cDNA clones encode DTP4polypeptides.

Example 9 Preparation of a Plant Expression Vector Containing a Homologto the Arabidopsis Lead Gene

Sequences homologous to the Arabidopsis AT-DTP4 polypeptide can beidentified using sequence comparison algorithms such as BLAST (BasicLocal Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403-410(1993); see also the explanation of the BLAST algorithm on the worldwide web site for the National Center for Biotechnology Information atthe National Library of Medicine of the National Institutes of Health).Sequences encoding homologous DTP4 polypeptides can be PCR-amplified byany of the following methods.

Method 1 (RNA-based): If the 5′ and 3′ sequence information for theprotein-coding region, or the 5′ and 3′ UTR, of a gene encoding a DTP4polypeptide homolog is available, gene-specific primers can be designedas outlined in Example 5. RT-PCR can be used with plant RNA to obtain anucleic acid fragment containing the protein-coding region flanked byattB1 (SEQ ID NO:10) and attB2 (SEQ ID NO:11) sequences. The primer maycontain a consensus Kozak sequence (CAACA) upstream of the start codon.

Method 2 (DNA-based): Alternatively, if a cDNA clone is available for agene encoding a DTP4 polypeptide homolog, the entire cDNA insert(containing 5′ and 3′ non-coding regions) can be PCR amplified. Forwardand reverse primers can be designed that contain either the attB1sequence and vector-specific sequence that precedes the cDNA insert orthe attB2 sequence and vector-specific sequence that follows the cDNAinsert, respectively. For a cDNA insert cloned into the vectorpBulescript SK+, the forward primer VC062 (SEQ ID NO:14) and the reverseprimer VC063 (SEQ ID NO:15) can be used.

Method 3 (genomic DNA): Genomic sequences can be obtained using longrange genomic PCR capture. Primers can be designed based on the sequenceof the genomic locus and the resulting PCR product can be sequenced. Thesequence can be analyzed using the FGENESH (Salamov, A. and Solovyev, V.(2000) Genome Res., 10: 516-522) program, and optionally, can be alignedwith homologous sequences from other species to assist in identificationof putative introns.

The above methods can be modified according to procedures known by oneskilled in the art. For example, the primers of Method 1 may containrestriction sites instead of attB1 and attB2 sites, for subsequentcloning of the PCR product into a vector containing attB1 and attB2sites. Additionally, Method 2 can involve amplification from a cDNAclone, a lambda clone, a BAC clone or genomic DNA.

A PCR product obtained by either method above can be combined with theGATEWAY® donor vector, such as pDONR™/Zeo (INVITROGEN™) or pDONR™ 221(INVITROGEN™), using a BP Recombination Reaction. This process removesthe bacteria lethal ccdB gene, as well as the chloramphenicol resistancegene (CAM) from pDONR™ 221 and directionally clones the PCR product withflanking attB1 and attB2 sites to create an entry clone. Using theINVITROGEN™ GATEWAY® CLONASE™ technology, the sequence encoding thehomologous DTP4 polypeptide from the entry clone can then be transferredto a suitable destination vector, such as pBC-Yellow, PHP27840 orPHP23236 (PCT Publication No. WO/2012/058528; herein incorporated byreference), to obtain a plant expression vector for use withArabidopsis, soybean and corn, respectively.

Sequences of the attP1 and attP2 sites of donor vectors pDONR™/Zeo orpDONR™ 221 are given in SEQ ID NOs:2 and 3, respectively. The sequencesof the attR1 and attR2 sites of destination vectors pBC-Yellow, PHP27840and PHP23236 are given in SEQ ID NOs:8 and 9, respectively. A BPReaction is a recombination reaction between an Expression Clone (or anattB-flanked PCR product) and a Donor (e.g., pDONR™) Vector to create anEntry Clone. A LR Reaction is a recombination between an Entry Clone anda Destination Vector to create an Expression Clone. A Donor Vectorcontains attP1 and attP2 sites. An Entry Clone contains attL1 and attL2sites (SEQ ID NOs:4 and 5, respectively). A Destination Vector containsattR1 and attR2 site. An Expression Clone contains attB1 and attB2sites. The attB1 site is composed of parts of the attL1 and attR1 sites.The attB2 site is composed of parts of the attL2 and attR2 sites.

Alternatively a MultiSite GATEWAY® LR recombination reaction betweenmultiple entry clones and a suitable destination vector can be performedto create an expression vector.

Example 10 Preparation of Soybean Expression Vectors and Transformationof Soybean with Validated Arabidopsis Lead Genes

Soybean plants can be transformed to overexpress a validated Arabidopsislead gene or the corresponding homologs from various species in order toexamine the resulting phenotype.

The same GATEWAY® entry clone described in Example 5 can be used todirectionally clone each gene into the PHP27840 vector (PCT PublicationNo. WO/2012/058528) such that expression of the gene is under control ofthe SCP1 promoter (International Publication No. 03/033651).

Soybean embryos may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. Techniques forsoybean transformation and regeneration have been described inInternational Patent Publication WO 2009/006276, the contents of whichare herein incorporated by reference.

T1 plants can be subjected to a soil-based drought stress. Using imageanalysis, plant area, volume, growth rate and color analysis can betaken at multiple times before and during drought stress. Overexpressionconstructs that result in a significant delay in wilting or leaf areareduction, yellow color accumulation and/or increased growth rate duringdrought stress will be considered evidence that the Arabidopsis genefunctions in soybean to enhance drought tolerance.

Soybean plants transformed with validated genes can then be assayedunder more vigorous field-based studies to study yield enhancementand/or stability under well-watered and water-limiting conditions.

Example 11 Transformation of Maize with Validated Arabidopsis Lead GenesUsing Particle Bombardment

Maize plants can be transformed to overexpress a validated Arabidopsislead gene or the corresponding homologs from various species in order toexamine the resulting phenotype.

The same GATEWAY® entry clone described in Example 5 can be used todirectionally clone each gene into a maize transformation vector.Expression of the gene in the maize transformation vector can be undercontrol of a constitutive promoter such as the maize ubiquitin promoter(Christensen et al., (1989) Plant Mol. Biol. 12:619-632 and Christensenet al., (1992) Plant Mol. Biol. 18:675-689)

The recombinant DNA construct described above can then be introducedinto corn cells by particle bombardment. Techniques for corntransformation by particle bombardment have been described inInternational Patent Publication WO 2009/006276, the contents of whichare herein incorporated by reference.

T1 plants can be subjected to a soil-based drought stress. Using imageanalysis, plant area, volume, growth rate and color analysis can betaken at multiple times before and during drought stress. Overexpressionconstructs that result in a significant delay in wilting or leaf areareduction, yellow color accumulation and/or increased growth rate duringdrought stress will be considered evidence that the Arabidopsis genefunctions in maize to enhance drought tolerance.

Example 12 Electroporation of Agrobacterium tumefaciens LBA4404

Electroporation competent cells (40 μL), such as Agrobacteriumtumefaciens LBA4404 containing PHP10523 (PCT Publication No.WO/2012/058528), are thawed on ice (20-30 min). PHP10523 contains VIRgenes for T-DNA transfer, an Agrobacterium low copy number plasmidorigin of replication, a tetracycline resistance gene, and a Cos sitefor in vivo DNA bimolecular recombination. Meanwhile the electroporationcuvette is chilled on ice. The electroporator settings are adjusted to2.1 kV. A DNA aliquot (0.5 μL parental DNA at a concentration of 0.2μg-1.0 μg in low salt buffer or twice distilled H₂O) is mixed with thethawed Agrobacterium tumefaciens LBA4404 cells while still on ice. Themixture is transferred to the bottom of electroporation cuvette and keptat rest on ice for 1-2 min. The cells are electroporated (Eppendorfelectroporator 2510) by pushing the “pulse” button twice (ideallyachieving a 4.0 millisecond pulse). Subsequently, 0.5 mL of roomtemperature 2×YT medium (or SOC medium) are added to the cuvette andtransferred to a 15 mL snap-cap tube (e.g., FALCON™ tube). The cells areincubated at 28-30° C., 200-250 rpm for 3 h.

Aliquots of 250 μL are spread onto plates containing YM medium and 50μg/mL spectinomycin and incubated three days at 28-30° C. To increasethe number of transformants one of two optional steps can be performed:

Option 1: Overlay plates with 30 μL of 15 mg/mL rifampicin. LBA4404 hasa chromosomal resistance gene for rifampicin. This additional selectioneliminates some contaminating colonies observed when using poorerpreparations of LBA4404 competent cells.

Option 2: Perform two replicates of the electroporation to compensatefor poorer electrocompetent cells.

Identification of Transformants:

Four independent colonies are picked and streaked on plates containingAB minimal medium and 50 μg/mL spectinomycin for isolation of singlecolonies. The plates are incubated at 28° C. for two to three days. Asingle colony for each putative co-integrate is picked and inoculatedwith 4 mL of 10 g/L bactopeptone, 10 g/L yeast extract, 5 g/L sodiumchloride and 50 mg/L spectinomycin. The mixture is incubated for 24 h at28° C. with shaking. Plasmid DNA from 4 mL of culture is isolated usingQiagen® Miniprep and an optional Buffer PB wash. The DNA is eluted in 30μL. Aliquots of 2 L are used to electroporate 20 L of DH10b+20 L oftwice distilled H₂O as per above. Optionally a 15 L aliquot can be usedto transform 75-100 μL of INVITROGEN™ Library Efficiency DH5α. The cellsare spread on plates containing LB medium and 50 μg/mL spectinomycin andincubated at 37° C. overnight.

Three to four independent colonies are picked for each putativeco-integrate and inoculated 4 mL of 2×YT medium (10 g/L bactopeptone, 10g/L yeast extract, 5 g/L sodium chloride) with 50 μg/mL spectinomycin.The cells are incubated at 37° C. overnight with shaking. Next, isolatethe plasmid DNA from 4 mL of culture using QIAprep® Miniprep withoptional Buffer PB wash (elute in 50 μL). Use 8 L for digestion withSail (using parental DNA and PHP10523 as controls). Three moredigestions using restriction enzymes BamHI, EcoRI, and HindIII areperformed for 4 plasmids that represent 2 putative co-integrates withcorrect Sail digestion pattern (using parental DNA and PHP10523 ascontrols). Electronic gels are recommended for comparison.

Example 13 Transformation of Maize Using Agrobacterium

Maize plants can be transformed to overexpress a validated Arabidopsislead gene or the corresponding homologs from various species in order toexamine the resulting phenotype.

Agrobacterium-mediated transformation of maize is performed essentiallyas described by Zhao et al. in Meth. Mol. Biol. 318:315-323 (2006) (seealso Zhao et al., Mol. Breed. 8:323-333 (2001) and U.S. Pat. No.5,981,840 issued Nov. 9, 1999, incorporated herein by reference). Thetransformation process involves bacterium innoculation, co-cultivation,resting, selection and plant regeneration.

1. Immature Embryo Preparation:

Immature maize embryos are dissected from caryopses and placed in a 2 mLmicrotube containing 2 mL PHI-A medium.

2. Agrobacterium Infection and Co-Cultivation of Immature Embryos:

2.1 Infection Step:

PHI-A medium of (1) is removed with 1 mL micropipettor, and 1 mL ofAgrobacterium suspension is added. The tube is gently inverted to mix.The mixture is incubated for 5 min at room temperature.

2.2 Co-Culture Step:

The Agrobacterium suspension is removed from the infection step with a 1mL micropipettor. Using a sterile spatula the embryos are scraped fromthe tube and transferred to a plate of PHI-B medium in a 100×15 mm Petridish. The embryos are oriented with the embryonic axis down on thesurface of the medium. Plates with the embryos are cultured at 20° C.,in darkness, for three days. L-Cysteine can be used in theco-cultivation phase. With the standard binary vector, theco-cultivation medium supplied with 100-400 mg/L L-cysteine is criticalfor recovering stable transgenic events.

3. Selection of Putative Transgenic Events:

To each plate of PHI-D medium in a 100×15 mm Petri dish, 10 embryos aretransferred, maintaining orientation and the dishes are sealed withparafilm. The plates are incubated in darkness at 28° C. Activelygrowing putative events, as pale yellow embryonic tissue, are expectedto be visible in six to eight weeks. Embryos that produce no events maybe brown and necrotic, and little friable tissue growth is evident.Putative transgenic embryonic tissue is subcultured to fresh PHI-Dplates at two-three week intervals, depending on growth rate. The eventsare recorded.

4. Regeneration of T0 Plants:

Embryonic tissue propagated on PHI-D medium is subcultured to PHI-Emedium (somatic embryo maturation medium), in 100×25 mm Petri dishes andincubated at 28° C., in darkness, until somatic embryos mature, forabout ten to eighteen days. Individual, matured somatic embryos withwell-defined scutellum and coleoptile are transferred to PHI-F embryogermination medium and incubated at 28° C. in the light (about 80 μEfrom cool white or equivalent fluorescent lamps). In seven to ten days,regenerated plants, about 10 cm tall, are potted in horticultural mixand hardened-off using standard horticultural methods.

Media for Plant Transformation:

-   -   1. PHI-A: 4 g/L CHU basal salts, 1.0 mL/L 1000× Eriksson's        vitamin mix, 0.5 mg/L thiamin HCl, 1.5 mg/L 2,4-D, 0.69 g/L        L-proline, 68.5 g/L sucrose, 36 g/L glucose, pH 5.2. Add 100 μM        acetosyringone (filter-sterilized).    -   2. PHI-B: PHI-A without glucose, increase 2,4-D to 2 mg/L,        reduce sucrose to 30 g/L and supplemented with 0.85 mg/L silver        nitrate (filter-sterilized), 3.0 g/L Gelrite®, 100 μM        acetosyringone (filter-sterilized), pH 5.8.    -   3. PHI-C: PHI-B without Gelrite® and acetosyringonee, reduce        2,4-D to 1.5 mg/L and supplemented with 8.0 g/L agar, 0.5 g/L        2-[N-morpholino]ethane-sulfonic acid (MES) buffer, 100 mg/L        carbenicillin (filter-sterilized).    -   4. PHI-D: PHI-C supplemented with 3 mg/L bialaphos        (filter-sterilized).    -   5. PHI-E: 4.3 g/L of Murashige and Skoog (MS) salts, (Gibco, BRL        11117-074), 0.5 mg/L nicotinic acid, 0.1 mg/L thiamine HCl, 0.5        mg/L pyridoxine HCl, 2.0 mg/L glycine, 0.1 g/L myo-inositol, 0.5        mg/L zeatin (Sigma, Cat. No. Z-0164), 1 mg/L indole acetic acid        (IAA), 26.4 μg/L abscisic acid (ABA), 60 g/L sucrose, 3 mg/L        bialaphos (filter-sterilized), 100 mg/L carbenicillin        (filter-sterilized), 8 g/L agar, pH 5.6.    -   6. PHI-F: PHI-E without zeatin, IAA, ABA; reduce sucrose to 40        g/L; replacing agar with 1.5 g/L Gelrite®; pH 5.6.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al., Bio/Technology 8:833-839 (1990)).

Transgenic T0 plants can be regenerated and their phenotype determined.T1 seed can be collected.

Furthermore, a recombinant DNA construct containing a validatedArabidopsis gene can be introduced into an elite maize inbred lineeither by direct transformation or introgression from a separatelytransformed line.

Transgenic plants, either inbred or hybrid, can undergo more vigorousfield-based experiments to study yield enhancement and/or stabilityunder water limiting and water non-limiting conditions.

Subsequent yield analysis can be done to determine whether plants thatcontain the validated Arabidopsis lead gene have an improvement in yieldperformance (under water limiting or non-limiting conditions), whencompared to the control (or reference) plants that do not contain thevalidated Arabidopsis lead gene. Specifically, water limiting conditionscan be imposed during the flowering and/or grain fill period for plantsthat contain the validated Arabidopsis lead gene and the control plants.Plants containing the validated Arabidopsis lead gene would have lessyield loss relative to the control plants, for example, at least 25%, atleast 20%, at least 15%, at least 10% or at least 5% less yield loss,under water limiting conditions, or would have increased yield, forexample, at least 5%, at least 10%, at least 15%, at least 20% or atleast 25% increased yield, relative to the control plants under waternon-limiting conditions.

Example 14A Preparation of Arabidopsis Lead Gene (At5g62180) ExpressionVector for Transformation of Maize

Using INVITROGEN™ GATEWAY® technology, an LR Recombination Reaction wasperformed to create the precursor plasmid pEV-DTP4. The vector pEV-DTP4contains the following expression cassette:

Ubiquitin promoter::At5g62180(SEQ ID NO:17)::PinII terminator; cassetteoverexpressing the gene of interest, Arabidopsis DTP4 polypeptide.

The At5g62180 sequence with alternative codons, SEQ ID NO:19, was alsocloned to create the precursor plasmid pEV-DTP4ac, which contains thefollowing expression cassette: Ubiquitin promoter::At5g62180 (SEQ IDNO:19)::SB-GKAF terminator; cassette overexpressing the gene ofinterest, Arabidopsis DTP4 polypeptide.

The SB-GKAF terminator is described in U.S. application Ser. No.14/236,499, herein incorporated by reference.

Example 14B Transformation of Maize with the Arabidopsis Lead Gene(At5q62180) Using Agrobacterium

The DTP4 polypeptide expression cassette present in vector pEV-DTP4, andthe DTP4 polypeptide expression cassette present in vector pEV-DTP4accan be introduced into a maize inbred line, or a transformable maizeline derived from an elite maize inbred line, usingAgrobacterium-mediated transformation as described in Examples 12 and13.

Vector pEV-DTP4 can be electroporated into the LBA4404 Agrobacteriumstrain containing vector PHP10523 (PCT Publication No. WO/2012/058528)to create the co-integrate vector pCV-DTP4. The co-integrate vector isformed by recombination of the 2 plasmids, pEV-DTP4 and PHP10523,through the COS recombination sites contained on each vector. Theco-integrate vector pCV-DTP4 contains the same expression cassette asabove (Example 14A) in addition to other genes (TET, TET, TRFA, ORIterminator, CTL, ORI V, VIR C1, VIR C2, VIR G, VIR B) needed for theAgrobacterium strain and the Agrobacterium-mediated transformation.

Similarly, the vector pEV-DTP4ac and PHP10523 were recombined to givethe co-integrate vector pCV-DTP4ac. The co-integrate vector pCV-DTP4accontains the same expression cassette as pEV-DTP4ac (Example 14A) inaddition to other genes (TET, TET, TRFA, ORI terminator, CTL, ORI V, VIRC1, VIR C2, VIR G, VIR B) needed for the Agrobacterium strain and theAgrobacterium-mediated transformation

Example 15 Preparation of the Destination Vector PHP23236 forTransformation into Gaspe Flint Derived Maize Lines

Destination vector PHP23236 was obtained by transformation ofAgrobacterium strain LBA4404 containing plasmid PHP10523 with plasmidPHP23235 and isolation of the resulting co-integration product. PlasmidsPHP23236, PHP10523 and PHP23235 are described in PCT Publication No.WO/2012/058528, herein incorporated by reference. Destination vectorPHP23236, can be used in a recombination reaction with an entry clone asdescribed in Example 16 to create a maize expression vector fortransformation of Gaspe Flint-derived maize lines.

Example 16 Preparation of Plasmids for Transformation into Gaspe FlintDerived Maize Lines

Using the INVITROGEN™ GATEWAY® LR Recombination technology, theprotein-coding region of the At5g62180 candidate gene, was directionallycloned into the destination vector PHP23236 (PCT Publication No.WO/2012/058528) to create an expression vector, pGF-DTP4. Thisexpression vector contains the protein-coding region of interest,encoding the DTP4 polypeptide, under control of the UBI promoter and isa T-DNA binary vector for Agrobacterium-mediated transformation intocorn as described, but not limited to, the examples described herein.

Example 17 Transformation of Gaspe Flint Derived Maize Lines with aValidated Arabidopsis Lead Gene

Maize plants can be transformed to overexpress the Arabidopsis lead geneor the corresponding homologs from other species in order to examine theresulting phenotype. Gaspe Flint derived maize lines can be transformedand analyzed as previously described in PCT Publication No.WO/2012/058528, the contents of which are herein incorporated byreference.

Example 18A Evaluation of Gaspe Flint Derived Maize Lines for DroughtTolerance

Transgenic Gaspe Flint derived maize lines containing the candidate genecan be screened for tolerance to drought stress in the following manner.

Transgenic maize plants are subjected to well-watered conditions(control) and to drought-stressed conditions. Transgenic maize plantsare screened at the T1 stage or later.

For plant growth, the soil mixture consists of ⅓ TURFACE®, ⅓ SB300 and ⅓sand. All pots are filled with the same amount of soil±10 grams. Potsare brought up to 100% field capacity (“FC”) by hand watering. Allplants are maintained at 60% FC using a 20-10-20 (N-P-K) 125 ppm Nnutrient solution. Throughout the experiment pH is monitored at leastthree times weekly for each table. Starting at 13 days after planting(DAP), the experiment can be divided into two treatment groups, wellwatered and reduce watered. All plants comprising the reduced wateredtreatment are maintained at 40% FC while plants in the well wateredtreatment are maintained at 80% FC. Reduced watered plants are grown for10 days under chronic drought stress conditions (40% FC). All plants areimaged daily throughout chronic stress period. Plants are sampled formetabolic profiling analyses at the end of chronic drought period, 22DAP. At the conclusion of the chronic stress period all plants areimaged and measured for chlorophyll fluorescence. Reduced watered plantsare subjected to a severe drought stress period followed by a recoveryperiod, 23-31 DAP and 32-34 DAP respectively. During the severe droughtstress, water and nutrients are withheld until the plants reached 8% FC.At the conclusion of severe stress and recovery periods all plants areagain imaged and measured for chlorophyll fluorescence. The probabilityof a greater Student's t Test is calculated for each transgenic meancompared to the appropriate null mean (either segregant null orconstruct null). A minimum (P<t) of 0.1 is used as a cut off for astatistically significant result.

Example 18B Evaluation of Maize Lines for Drought Tolerance

Lines with Enhanced Drought Tolerance can also be screened using thefollowing method (see also FIG. 3 for treatment schedule):

Transgenic maize seedlings are screened for drought tolerance bymeasuring chlorophyll fluorescence performance, biomass accumulation,and drought survival. Transgenic plants are compared against the nullplant (i.e., not containing the transgene). Experimental design is aRandomized Complete Block and Replication consist of 13 positive plantsfrom each event and a construct null (2 negatives each event).

Plant are grown at well watered (WW) conditions=60% Field Capacity (%FC) to a three leaf stage. At the three leaf stage and under WWconditions the first fluorescence measurement is taken on the uppermostfully extended leaf at the inflection point, in the leaf margin andavoiding the mid rib.

This is followed by imposing a moderate drought stress (FIG. 3, day 13,MOD DRT) by maintaining 20% FC for duration of 9 to 10 days. During thisstress treatment leaves may appear gray and rolling may occur. At theend of MOD DRT period, plants are recovered (MOD rec) by increasing to25% FC. During this time, leaves will begin to unroll. This is a timesensitive step that may take up to 1 hour to occur and can be dependentupon the construct and events being tested. When plants appear to haverecovered completed (leaves unrolled), the second fluorescencemeasurement is taken.

This is followed by imposing a severe drought stress (SEV DRT) bywithholding all water until the plants collapse. Duration of severedrought stress is 8-10 days and/or when plants have collapse.Thereafter, a recovery (REC) is imposed by watering all plants to 100%FC. Maintain 100% FC 72 hours. Survival score (yes/no) is recorded after24, 48 and 72 hour recovery.

The entire shoot (Fresh) is sampled and weights are recorded (Freshshoot weights). Fresh shoot material is then dried for 120 hrs at 70degrees at which time a Dry Shoot weight is recorded.

Measured variables are defined as follows:

The variable “Fv′/Fm′ no stress” is a measure of the optimum quantumyield (Fv′/Fm′) under optimal water conditions on the uppermost fullyextended leaf (most often the third leaf) at the inflection point, inthe leaf margin and avoiding the mid rib. Fv′/Fm′ provides an estimateof the maximum efficiency of PSII photochemistry at a given PPFD, whichis the PSII operating efficiency if all the PSII centers were open(Q_(A) oxidized).

The variable “Fv′/Fm′ stress” is a measure of the optimum quantum yield(Fv′/Fm′) under water stressed conditions (25% field capacity). Themeasure is preceded by a moderate drought period where field capacitydrops from 60% to 20%. At which time the field capacity is brought to25% and the measure collected.

The variable “phiPSII_no stress” is a measure of Photosystem II (PSII)efficiency under optimal water conditions on the uppermost fullyextended leaf (most often the third leaf) at the inflection point, inthe leaf margin and avoiding the mid rib. The phiPSII value provides anestimate of the PSII operating efficiency, which estimates theefficiency at which light absorbed by PSII is used for Q_(A) reduction.

The variable “phiPSII_stress” is a measure of Photosystem II (PSII)efficiency under water stressed conditions (25% field capacity). Themeasure is preceded by a moderate drought period where field capacitydrops from 60% to 20%. At which time the field capacity is brought to25% and the measure collected.

Example 19A Yield Analysis of Maize Lines with the Arabidopsis Lead Gene

A recombinant DNA construct containing a validated Arabidopsis gene canbe introduced into an elite maize inbred line either by directtransformation or introgression from a separately transformed line.

Transgenic plants either inbred or hybrid, can undergo more vigorousfield-based experiments to study yield enhancement and/or stabilityunder well-watered and water-limiting conditions.

Subsequent yield analysis can be done to determine whether plants thatcontain the validated Arabidopsis lead gene have an improvement in yieldperformance under water-limiting conditions, when compared to thecontrol plants that do not contain the validated Arabidopsis lead gene.Specifically, drought conditions can be imposed during the floweringand/or grain fill period for plants that contain the validatedArabidopsis lead gene and the control plants. Reduction in yield can bemeasured for both. Plants containing the validated Arabidopsis lead genehave less yield loss relative to the control plants, for example, atleast 25%, at least 20%, at least 15%, at least 10% or at least 5% lessyield loss.

The above method may be used to select transgenic plants with increasedyield, under water-limiting conditions and/or well-watered conditions,when compared to a control plant not comprising said recombinant DNAconstruct. Plants containing the validated Arabidopsis lead gene mayhave increased yield, under water-limiting conditions and/orwell-watered conditions, relative to the control plants, for example, atleast 5%, at least 10%, at least 15%, at least 20% or at least 25%increased yield.

Example 19B Yield Analysis of Maize Lines Transformed with pCV-DTP4Encoding the Arabidopsis Lead Gene At5g62180

Nine transgenic events were field tested at 3 locations, Locations “A”,“E”, and “B”. At the “B” location, drought conditions were imposedduring flowering (“B1”; flowering stress) and during the grain fillperiod (“B2”; grain fill stress). The “A” location was well-watered, andthe “E” location experienced mild drought during the grain-fillingperiod. Yield data (bushel/acre; bu/ac) of the 9 transgenic events isshown in FIG. 5 together with the wt and bulk null control (BN).Statistical significance is reported at P<0.1 for a two-tailed test.

The significant values (with p-value less than or equal to 0.1 with a2-tailed test) are shown in bold when the value is greater than the nullcomparator and in bold and italics when that value is less than thenull.

In the most severe “B2” location it was neutral. In an intermediate “B1”location three events were positive but the experiment was unreliablebecause of the unexpected divergence between null and wild typeperformance.

Example 19C Yield Analysis of Maize Lines Transformed with pCV-DTP4acEncoding the Arabidopsis Lead Gene At5q62180

First Year Testing:

The AT-DTP4 polypeptide (SEQ ID NO:18) encoded by the nucleotidesequence (SEQ ID NO:19) present in the vector pCV-DTP4ac was introducedinto a transformable maize line derived from an elite maize inbred lineas described in Examples 14A and 14B.

Eight transgenic events were field tested at 5 locations A, E, C, D, andB. At the location B, mild drought conditions were imposed duringflowering (this treatment was divided into 2 areas B1-a and B1-b) andsevere drought conditions were imposed during the grain fill period(“grain fill stress; B2). The “A” location was well-watered, and the “E”location experienced mild drought during the grain-filling period. Both“C” and “D” locations experienced severe stress (FIG. 10).

Yield data were collected in all locations, with 3-6 replicates perlocation.

Yield data (bushel/acre; bu/ac) for the 8 transgenic events is shown inFIGS. 10A and 10B together with the bulk null control (BN). Yieldanalysis was by ASREML (VSN International Ltd), and the values are BLUPs(Best Linear Unbiased Prediction) (Cullis, B. R et al (1998) Biometrics54: 1-18, Gilmour, A. R. et al (2009). ASReml User Guide 3.0, Gilmour,A. R., et al (1995) Biometrics 51: 1440-50).

As shown in FIG. 10A, consistent effect of the transgene on yield wasseen in at all the locations that resulted in a significant positiveeffect in 3-8 events., with the positive event magnitude ranging from 4to 16 bu/ac.

FIG. 10B shows the yield analysis by grouping locations into “highstress”, “low stress” and “no stress (TPE)” category. As can be seenfrom FIG. 15B, positive effect of the transgene on yield was seen forall 8 transgenic events in high stress and low stress locations, and in2 events in the “no stress category”.

Effect of the transgene on other agronomic characteristics were alsoevaluated; such as plant and ear height (EARHT, PLTHT; at location “A”(no-stress) and location “D” (high-stress) locations), thermal time toshed (TTSHED: locations “D” and B2-b (location B at grain fillingstress); both high-stress locations), percent root lodging or stalklodging (LRTLPC, STLPCT; at the location “E” (low stress location). Asshown in FIG. 11A and FIG. 11B, no effect of the transgene on thesecharacteristics was observed.

Second Year Testing:

The eight transgenic events field tested for the first year, were fieldtested for a second year multiple locations with different levels ofdrought stress: no stress (8 locations; 1-8 in FIG. 14A); medium stress(5 locations; 9-13 in FIG. 14A); and severe stress (5 locations; 14-18in FIG. 14A).

The eight transgenic events were also tested in three low nitrogenlocations (locations 19-21 in FIG. 14A)

Yield data were collected in all locations, with 3-6 replicates perlocation.

Yield data (bushel/acre; bu/ac) for the 8 transgenic events is shown inFIG. 14A-14C for the drought stress, and in FIG. 15 the yield data inresponse to low nitrogen is shown; all the data are shown with the bulknull control (BN). Yield analysis was by ASREML (VSN International Ltd),and the values are BLUPs (Best Linear Unbiased Prediction) (Cullis, B. Ret al (1998) Biometrics 54: 1-18, Gilmour, A. R. et al (2009). ASRemlUser Guide 3.0, Gilmour, A. R., et al (1995) Biometrics 51: 1440-50).FIG. 14D shows the multi-location anlaysis for the “no stress”, “mediumstress” and “severe stress” locations, along with the multi-locationanalysis for all the drought stress locations.

As shown in FIG. 14A-FIG. 14D, effect of the transgene on yield was seenin at least one location with no stress, at least 2 locations in mediumand severe stress; the multi-location analysis in FIG. 14D showsconsistent positive effect of the transgene on yield., with the positiveevent magnitude ranging from 15 to 20 bu/ac, under medium stress.

FIG. 14D shows the yield analysis by grouping locations into “highstress”, “low stress” and “no stress” category. As can be seen from FIG.14B, positive effect of the transgene on yield was seen for all 8transgenic events in medium stress and severe stress locations, and in 2events in the “no stress category”.

As shown in FIG. 15, no positive effect of the transgene on yield wasobserved under low nitrogen conditions.

Example 19D Yield Analysis of Maize Lines Transformed with pCV-AT-CXE8acEncoding the Arabidopsis DTP4 Homolog AT-CXE8

The AT-CXE8 polypeptide (SEQ ID NO:64) encoded by the nucleotidesequence (SEQ ID NO:63), with alternative codons, was cloned asdescribed in Example 14A and Example 14B; using the Invitrogen Gatewaytechnology.

The At2g45600 sequence with alternative codons, SEQ ID NO:63 was alsocloned to create the precursor plasmid pEV-CXE8ac, which contains thefollowing expression cassette: Zm Ubiquitin promoter::At2g45600 (SEQ IDNO:63)::Sb-Ubi terminator; cassette overexpressing the gene of interest,the AT-DTP4 homolog, Arabidopsis CXE8 polypeptide.

The AT-CXE8 polypeptide (SEQ ID NO:64) encoded by the nucleotidesequence (SEQ ID NO:63) present in the vector pCV-AT-CXE8ac wasintroduced into a transformable maize line derived from an elite maizeinbred line as described in Examples 14A and 14B.

Seven transgenic events were field tested at 7 locations.

The seven transgenic events were field tested at multiple locations withdifferent levels of drought stress: no stress (1 location; location 28in FIG. 16A); medium stress (1 location; location 22 in FIG. 16A); andsevere stress (4 locations; locations 24-27 in FIG. 16A).

Yield data were collected in all locations, with 3-6 replicates perlocation.

Yield data (bushel/acre; bu/ac) for the seven transgenic events is shownin FIGS. 16A and 16B together with the bulk null control (BN). Yieldanalysis was by ASREML (VSN International Ltd), and the values are BLUPs(Best Linear Unbiased Prediction) (Cullis, B. R et al (1998) Biometrics54: 1-18, Gilmour, A. R. et al (2009). ASReml User Guide 3.0, Gilmour,A. R., et al (1995) Biometrics 51: 1440-50).

As shown in FIG. 16A, consistent effect of the transgene on yield wasseen at no stress and severe stress locations, that resulted in asignificant positive effect in 3-8 events, with the positive eventmagnitude ranging from 5 to 10 bu/ac.

FIG. 16B shows the yield analysis across locations, grouped by droughtstress levels. As can be seen from FIG. 16B, positive effect of thetransgene on yield was seen for 6 transgenic events in across locationanalysis, after taking all stress level locations together.

Example 20A Preparation of Maize DTP4 Polypeptide Lead Gene ExpressionVector for Transformation of Maize

The protein-coding region of the maize DTP4 homologs disclosed in theapplication can be introduced into the INVITROGEN™ vector pENTR/D-TOPO®to create entry clones.

Using INVITROGEN™ GATEWAY® technology, LR Recombination Reaction can beperformed with the entry clones and a destination vector to createprecursor plasmids. These vectors contain the following expressioncassette:

Ubiquitin promoter::Zm-DTP4-Polypeptide::Pin II terminator; cassetteoverexpressing the gene of interest.

Example 20B Transformation of Maize with Maize DTP4 Polypeptide LeadGene Using Agrobacterium

The maize DTP4 polypeptide expression cassette present in the vectorsfrom the above example can be introduced into a maize inbred line, or atransformable maize line derived from an elite maize inbred line, usingAgrobacterium-mediated transformation as described in Examples 12 and13.

Any or of these vectors can be electroporated into the LBA4404Agrobacterium strain containing vector PHP10523 (PCT Publication No.WO/2012/058528) to create a co-integrate vector. The co-integrate vectoris formed by recombination of the 2 plasmids, the precursor plasmid andPHP10523, through the COS recombination sites contained on each vector.The co-integrate vector contains the same 3 expression cassettes asabove (Example 20A) in addition to other genes (TET, TET, TRFA, ORIterminator, CTL, ORI V, VIR C1, VIR C2, VIR G, VIR B) needed for theAgrobacterium strain and the Agrobacterium-mediated transformation.

Example 21 Preparation of Maize Expression Plasmids for Transformationinto Gaspe Flint Derived Maize Lines

Using the INVITROGEN™ GATEWAY® Recombination technology described inExample 9, the clones encoding maize DTP4 polypeptide homologs disclosedherein can be directionally cloned into the destination vector PHP23236(PCT Publication No. WO/2012/058528) to create expression vectors. Eachexpression vector contains the cDNA of interest under control of the UBIpromoter and is a T-DNA binary vector for Agrobacterium-mediatedtransformation into corn as described, but not limited to, the examplesdescribed herein.

Example 22 Transformation and Evaluation of Soybean with SoybeanHomologs of Validated Lead Genes

Based on homology searches, one or several candidate soybean homologs ofvalidated Arabidopsis lead genes can be identified and also be assessedfor their ability to enhance drought tolerance in soybean. Vectorconstruction, plant transformation and phenotypic analysis will besimilar to that in previously described Examples.

Example 23 Transformation of Arabidopsis with Maize and Soybean Homologsof Validated Lead Genes

Soybean and maize homologs to validated Arabidopsis lead genes can betransformed into Arabidopsis under control of the 35S promoter andassessed for their ability to enhance drought tolerance in Arabidopsis.Vector construction, plant transformation and phenotypic analysis willbe similar to that in previously described Examples.

Example 24 Transformation of Arabidopsis with DTP4 Polypeptides fromOther Species

Any of the DTP4 polypeptides disclosed herein, including the ones givenin Table 1 or Table 2, can be transformed into Arabidopsis under controlof the 35S promoter and assessed for their ability to enhance droughttolerance, or in any of the other assays described herein, inArabidopsis. Vector construction, plant transformation and phenotypicanalysis will be similar to that in previously described Examples.

Example 25A Osmotic Stress Assay

To assay the osmotic stress tolerance of a transgenic line, acombination of osmolytes in the media, such as water soluble inorganicsalts, sugar alcohols and high molecular weight non-penetratingosmolytes can be used to select for osmotically-tolerant plant lines.

The osmotic stress agents used in this quad stress assay are:

-   -   1) NaCl (sodium chloride)    -   2) Sorbitol    -   3) Mannitol    -   4) Polyethylene Glycol (PEG)        By providing these agents in the media, we aim to mimic multiple        stress conditions in the in vitro environment thereby giving the        plant the opportunity to respond to four stress agents.

Methods and Materials:

As there are four stress agents being used together, a quarter of eachtogether in a solution will denote 100% stress or an osmotic pressure of1.23 MPa. Therefore the following concentrations of each component areused in 100% quad media.

Stress agents Concentrations NaCl— 62.5 mM  Sorbitol- 125 mM Mannitol-125 mM PEG- 10%

Assay Conditions:

Seeds are surface sterilized and stratified for 48 hrs. About 100 seedsare inoculated in one plate and cultured in a growth chamber programmedfor 16 h of light at 22′C temperature and 50% relative humidity.Germination is scored as the emergence of radicle.

Assay Plan:

A 6-day assay and an extended 10-day assay are done to test the seedstransgenic Arabidopsis line for osmotic stress tolerance.

Day 0—Surface sterilized seeds of different drought leads and stratifyDay 2—Inoculated onto quad mediaDay 4—Counted for germination (48 hrs)Day 5—Counted for germination (72 hrs) I Take pictures or Scan platesfrom 48 hrs to 96 hrs.Day 6—Counted for germination (96 hrs)For the extended 10-day assay, germination is scored from 48 hrs to 96hrs. On day 7, 8, 9 and 10, the emerged seedlings were checked forgreenness and four leaf stage.

Preparation of Media:

Germination medium (GM or 0% quad media) for 1 liter:

MS salt 4.3 g Sucrose 10 g 1000x Vitamin mix 1 ml MES (pH 5.7 with KOH)10 ml Phytagel (0.3%) 3 gTo this the quad agents (the four osmolytes) are added by individuallyweighing the specific amounts in grams for their respectiveconcentrations. Quad media preparation chart for all concentrations ofosmolytes is given in Table 6.

TABLE 6 Quad Media Preparation Chart 10% 20% 30% 40% 50% 60% 70% 80% 90%100% NaCl 0.36 0.731 1.09 1.46 1.82 2.19 2.55 2.9 3.29 3.656 Mannitol2.27 4.55 8.83 9.1 11.38 13.66 15.93 18.2 20.49 22.77 Sorbitol 2.27 4.556.83 9.1 11.38 13.66 15.93 18.2 20.49 22.77 PEG 10 20 30 40 50 60 70 8090 100

Sterilization of Seeds:

Approximately 100 μl of Arabidopsis Columbia wild type seeds (col wt)and the seeds of the transgenic line to be tested are taken in 1.75 mlmicrofuge tubes and sterilized in ethanol for 1 min 30 sec followed byone wash with sterile water. Then they are subjected to bleach treatment(4% bleach with Tween 20) for 2 min 30 sec. This is followed by 4 to 5washes in sterile water. Seeds are stratified at 4° C. for 48 hrs beforeinoculation.

Inoculation of Seeds:

Stratified seeds are plated onto a single plate of each quad stressconcentration as given in Table 6. Plates are cultured in the chambersset at 16 h of light at 22° C. temperature and 50% relative humidity.Germination is scored as the emergence of radicle over a period of 48 to96 hrs. Seeds are counted manually using a magnifying lens. Plates arescanned at 800 dpi using Epson scanner 10,000 XL and photographed. Incase of the extended assay, leaf greenness (manual) and true leafemergence i.e, 4Leaf stage (manual scoring) are also scored over aperiod of 10 days to account for the growth rate and health of thegerminated seedlings.

The data is analyzed as percentage germination to the total number ofseeds that are inoculated. Analyzed data is represented in the form ofbar graphs and sigmoid curves by plotting quad concentrations againstpercent germination.

Example 25B Seedling Emergence Under Osmotic Stress of TransgenicArabidopsis Seeds with AT-DTP4 Proteins

T1 seeds from transgenic Arabidopsis line with AT-DTP4 protein,containing the 35S promoter::At5g62180 expression constructpBC-Yellow-At5g62180, generated as described above, were tested forseedling emergence under osmotic stress as described in Example 25A.

Arabidopsis Columbia seeds were used as wild-type control and at 60%there was a dip in germination and thereafter a decline and zerogermination at 100%, as shown in Table 7.

Table 7 presents the percentage germination data at 48 hours forseedling emergence under osmotic stress.

TABLE 7 Percentage Germination Data in Arabidopsis Quad % in the %Germination % Germination for Media for WT At5g62180 0 96 90 10 80 87 2076 90 30 69 92 40 52 90 50 29 82 60 20 66 70 10 54 80 2 9 90 6 65 100 02

Seedling Emergence Under Osmotic Stress—10 Day Assay:

The results in Table 7 demonstrate that the transgenic Arabidopsis line(Line ID 64) containing the 35S promoter::At5g62180 expressionconstruct, pBC-Yellow-At5g62180, which was previously selected as havinga drought tolerance and ABA-hypersensitivity phenotype, alsodemonstrates increased seedling emergence under osmotic stress.

The osmotic stress assay for Line ID 64 was repeated, and scored forpercentage greenness and percentage leaf emergence in an extended 10 dayassay as well. The line was scored at 0% (GM or growth media), 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% quad, for germination at 48hours, and for percentage greenness and percentage leaf emergence in anextended 10 day assay. The results are shown in FIG. 6A and FIG. 6B.

Percentage greenness and percentage leaf emergence were assayed.Percentage greenness was scored as the percentage of seedlings withgreen leaves (cotyledonary or true leaves) compared to yellow, brown orpurple leaves. Greenness was scored manually and if there was any yellowor brown streaks on any of the 4 leaves, it was not considered green.Greenness was counted for seedlings with total green leaves only.

The leaf emergence was scored as the appearance of fully expanded leaves1 and 2, after the two cotyledonary leaves had fully expanded.Therefore, the percentage leaf emergence is the number of seedlings with2 true leaves or 4 leaves in total (2 cotyledonary and 2 true leaves).

The percentage germination experiment at 48 hours was repeated once morewith bulked seeds, in triplicates, and the results are shown in FIG. 7.Seeds were plated on MSO plate containing MS media+methioninesulphoximine and selected plants transplanted to the soil, seedsharvested and assayed.

Example 26A ABA/Root Growth Assay

Plants being sessile have evolved a higher adaptability to overcomeadverse environmental challenges. The phytohormone abscisic acid (ABA)is a key endogenous messenger in plants' responses to such stresses andtherefore understanding ABA signaling is essential for improving plantperformance especially under drought stress. Drought is a verycomplicated phenomenon involving several key regulators and in order tocapture wide spectrum of such players a multi-assay approach isimperative. A root growth assay has been developed keeping thisobjective in mind.

In the ABA/Root assay, the sensitivity of root growth on mediacontaining ABA post germination on MS media is used as the assaycriterion. MS media comprises of MS basal salts, MS vitamins, sucroseand phytagel as a gelling agent. ABA/Root assay will enable us topotentially capture both hypersensitive and hyposensitive outliers/leadsmaking it a powerful tool for screening of new genes and as a crossvalidation assay.

The ABA/Root assay is a two phase assay. Phase I includes growing seedson plain germination/MS media vertically under 230 μMol light intensity.After 5 days of germination, seedlings are picked and transferred tomedia comprising ABA. The position of the root tip at the time oftransfer is marked. The seedlings are allowed to grow vertically for 7days on media containing ABA with daily rotation of plates such thateach plate receives uniform light. On the seventh day, the plates areimaged and root phenotypes are analyzed. The overall schematic of theassay is presented in FIG. 8.

Example 26B ABA/Root Growth Assay with Transgenic Arabidopsis Seeds withAT-DTP4 Polypeptide

In this assay, an ABA hypersensitive outlier would be expected to haveseedlings arrested at the point of transfer whereas in an ABAhyposensitive outlier the roots would continue to grow because of theirinability to sense ABA in the media. For lines that are insensitive,would be expected to behave similar to WT, which would be the negativecontrol.

Assay Conditions:

WT seeds and transgenic seeds containing the pBC-yellow-At5g62180construct described in Example 5A were used for this assay. Seeds weresurface sterilized first with 100% ethanol followed with bleach+Tween 20solution followed by 4 washes of sterile water and stratified for 48hrs. Two rows of around 30 stratified seeds each were sown ongermination media and the plates were kept vertically in the growthchamber for 5 days. The growth chamber settings were 16 h of 230 μMollight at 22° C. temperature and 50% relative humidity. After 5 days, theseedlings were picked one by one and transferred to media containingdifferent concentrations of ABA, 0, 2.5, 5, 10, 15, 17.5, 20, 25 and 30μM ABA. The seedlings were grown vertically for 7 days. After 7 days,root phenotypes were analyzed and recorded. The representative resultsfor the concentrations in the range 15-25 μM are shown in FIG. 9.

Example 27 ABA Sensitivity Assay Percentage Germination Assay with DTP4Polypeptides in Arabidopsis

DTP4 polypeptides homologous to AT-DTP4 (SEQ ID NO:18) were tested fortheir ability to confer ABA-hypersensitivity by a percentage germinationassay as described in Example 7.

The cDNA protein-coding region for each of these homologs wassynthesized and cloned into the transformation vector. The homologs weretested for ABA hypersensitivity on 2 ABA concentrations, 1 μM and 2 μM.

Transgenic T2 seeds were selected, and used for the germination assay asdescribed in Example 7. Two Sesbania bispinosa homologssesgr1n.pk107.c11 and sesgr1n.pk079.h12 and (SEQ ID NOS:44 and 46,respectively), showed ABA hypersensitivity when they were directlyexpressed by the 35S promoter.

At 1 μM ABA, wild-type col-0 plants had >90% of germination rate at Day5. The transgenic line with AtDTP4 construct showed <90% germination onDay 5, as shown in FIG. 12A. The line with a construct expressing theDTP4 homologs sesgr1n.pk079.h12 (SEQ ID NO:47) showed about 70%germination, and that expressing the DTP4 homolog sesgr1n.pk107.c11 (SEQID NO:45) showed about 80% germination on day 3.

At 2 μM ABA, wild-type col-0 plants had >90% of germination rate at Day5. The transgenic line with AtDTP4 construct showed <70% germination onDay 5, as shown in FIG. 12B. The line with a construct expressing theDTP4 homolog sesgr1n.pk079.h12 (SEQ ID NO:47) showed <50% germination,and that expressing the DTP4 homolog sesgr1n.pk107.c11 (SEQ ID NO:45)showed <70% germination on day 5.

FIG. 12C shows the percentage germination assay for transgenicArabidopsis plants expressing some of the other DTP4 homologs that weretested, given in Table 9 and Table 10, respectively.

Example 28 ABA Sensitivity Assay Green Cotyledon Assay with DTP4Polypeptides in Arabidopsis

The DTP4 polypeptides given in Table 8 and Table 9 were tested for theirability to confer ABA hypersensitivity by a percentage green cotyledonassay as described below.

The cDNA protein-coding region for each of these homologs wassynthesized and cloned into the transformation vector. The homologs weretested for ABA hypersensitivity on 2 μM ABA containing medium.

Assay Conditions:

Seeds were surface sterilized and stratified for 96 hrs. About 100 seedswere inoculated in one plate and stratified for 96 hrs, then cultured ina growth chamber programmed for 16 h of light at 22° C. temperature and50% relative humidity. Seedlings with green cotyledons were scored.

Observations and Results:

Seedlings with green and expanded cotyledons ware scored in ½ MS mediaand 2 μM ABA on Day 5-7. Seeds were counted manually using a magnifyinglens. The data was analyzed as percentage seedlings with greencotyledons to the total number of seeds that were inoculated. Wild-typecol-0 plants normally have ˜60-70% of seedlings with green cotyledons.The line with pBC-yellow-At5g62180 (AtDTP4 expression constructdescribed and some homologs had scores<45% in this assay.

FIG. 13 and FIG. 12C show the green cotyledon assay and percentagegermination assay results respectively (Example 27) for transgenicArabidopsis plants expressing some of the other DTP4 polypeptides thatwere tested, given in Table 8 and Table 9, respectively.

TABLE 8 ABA Sensitivity Assay with DTP4 Polypeptides PercentageCotyledon SEQ germination greening Clone ID ID NO Type assay assayATDTP4 18 Type II Positive Positive sesgr1n.pk117.j17 39 Type II NeutralNeutral sesgr1n.pk062.h8 43 Type II Neutral Neutral sesgr1n.pk107.c11 45Type II Positive Positive sesgr1n.pk079.h12 47 Type II Positive Positivearttr1n.pk125.i16 49 Type II neutral neutral arttr1n.pk029.e11 51 TypeII neutral neutral arttr1n.pk120.m9 55 Type II neutral neutralhengr1n.pk028.m4 59 Type II neutral neutral icegr1n.pk156.e13 61 Type IIneutral neutral pepgr1n.pk190.l24 95 Type II neutral neutralpepgr1n.pk082.c4 97 Type II neutral neutral hengr1n.pk014.d12 101 TypeII neutral neutral ecalgr1n.pk137.m22 103 Type II neutral neutralahgr1c.pk108.k16 107 Type II neutral neutral arttr1n.pk193.a17 111 TypeII neutral neutral arttr1n.pk090.l10 113 Type II neutral neutral At-cxe5627 Type III neutral Positive At-cxe8 64 Type II neutral PositiveAt-cxe9 65 Type II neutral Negative At-cxe17 628 Type VI neutralnegative At-cxe18 66 Type IV neutral negative

TABLE 9 ABA Sensitivity Assay with DTP4 Polypeptides ID in graph in FIG.12C SEQ and Percentage Cotyledon ID FIG. germination greening Clone IDNO 13 Type assay assay Thhalv10005595m 117 GS3 Type II Neutral PositiveBn-Bo 119 GS6 Type II Positive Positive B-ole-someBnap 121 GS8 Type IINeutral Neutral B-napus2-1 123 GS9 Type II Neutral Positive D7MLB3_Al127 GS1 Type II Positive Positive R0I9H0_Cr 129 GS2 Type II NeutralPositive R0EXR3_Cr 130 GS4 Type II Positive Positive M4F4A4_Bp 131 GS5Type II Positive Positive M4EKG1_Bp 132 GS7 Type II Neutral NeutralGSVIVT01010672001 135 GS10 Type II Neutral Neutral

Example 29A ABA Sensitivity Assay Root Architecture Assay in Arabidopsis

To test transgenic plants for alteration in root architecture inresponse to ABA, the root architecture assay is done as described inthis example.

Seeds are sterilized using 50% household bleach 0.01% Triton X-100solution and on petri plates containing the following medium: 0.5×N-FreeHoagland's, 8 mM KNO₃, 1% sucrose, 1 mM MES and 1% PHYTAGEL™,supplemented with 0.1 μM ABA, at a density of 4 seeds/plate. Typically10 plates are placed in a rack. Plates are kept for three days at 4° C.to stratify seeds and then held vertically for 12 days at 22° C. lightand 20° C. dark. Photoperiod is 16 h; 8 h dark, average light intensityis ˜180 μmol/m²/s. Racks (typically holding 10 plates each) are rotatedevery alternate day within each shelf. At day 12, plates are evaluatedfor seedling status, whole plate scan are taken, and analyzed for rootarea.

These seedlings grown on vertical plates are analyzed for root growthwith the software WINRHIZO® (Regent Instruments Inc), an image analysissystem specifically designed for root measurement. WINRHIZO® uses thecontrast in pixels to distinguish the light root from the darkerbackground. To identify the maximum amount of roots without picking upbackground, the pixel classification is kept at 150-170 and the filterfeature is used to remove objects that have a length/width ratio lessthan 10.0. The area on the plates analyzed is from the edge of theplant's leaves to about 1 cm from the bottom of the plate. The exactsame WINRHIZO® settings and area of analysis is used to analyze allplates within a batch. The total root length score given by WINRHIZO®for a plate is divided by the number of plants that have germinated andhave grown halfway down the plate. Eight plates for every line are grownand their scores are averaged. This average is then compared to theaverage of eight plates containing wild type seeds that have been grownat the same time.

Thirty seedlings from transgenic are compared to same number in controland probability value was generated. Transgenics with probability value(p-value) equal to and or more than E-03 is considered is validated inRA assay.

Example 29B Root Architecture Assay for Transgenic AT-DTP4 ArabidopsisPlants

The Arabidopsis DTP4 polypeptide gene (At5g62180; SEQ ID NO:16; NCBI GINo. 30697645) was tested for its ability to confer altered ABAsensitivity or in the following manner.

T3 seeds from seven single insertion events (named E3, E4, E5, E6, E7,E8 and E9) from transgenic Arabidopsis line with AT-DTP4 protein,containing the 35S promoter::At expression constructpBC-yellow-At5g62180, generated as described in Example 6, were testedfor alteration of root architecture due to presence of ABA in the media,as described in Example 27A.

Non-transformed Columbia seeds grown in the same conditions and at thesame time of the single insertion events served as a control. Singleline event and control seeds were subjected to the Root ArchitectureAssay, to test ABA sensitivity, following the procedure described inExample 29A.

Eight plates having 32 seedlings were scanned, and the pixel valuesobtained for each of the 32 roots of each event was compared with thepixel values obtained for the control.

T-test analysis was performed to show that the AT-DTP4 transgenic plantshave better root growth under 0.1 μM ABA, indicating altered ABAsensitivity as compared to the wt plants.

The p-value for different events, done as 2 different experiments on 2different days, is given in Table 10. The ones with probability value(p-value) equal to and or more than E-03 are shown in bold.

TABLE 10 P-values for RA Assay with AT-DTP4 Transgenic Plants Eventsttest (experiment 1) ttest (experiment 2) E3 5.77E−01 1.29E−01 E45.14E−04 4.69E−02 E5 6.36E−01  8.3E−01 E6 3.43E−07 1.11E−07 E7 2.08E−021.21E−01 E8 3.92E−04 3.12E−03 E9 8.22E−07 6.27E−05

Example 30 Detection of DTP4 Protein in Transgenic Maize Leaves by MassSpectrometry

The transgenic maize events from the two constructs used in the fieldyield trials described in Example 19 were regrown in a growth chamberuntil stage V5 to provide leaf samples for detection of DTP4 protein bymass spectrometry. Leaves were excised and ground in liquid nitrogen,and then the frozen powder was lyophilized. The protein from 10 mg oflyophilized leaf powder per sample was extracted and subjected toanalysis by mass spectrometry. AT-DTP4 protein was detected in all 8events of the pCV-DTP4ac construct.

Field grown transgenic events for construct pCV-DTP4ac were also usedfor DTP4 protein detection by the same mass spec method (FIG. 17). TheDTP4 protein was detected in V9 leaves of all transgenic events, but notin leaves of null plants. The greatest amount of DTP4 protein in thefield grown plants was detected in event DTP4-L17, as was observed withthe data for growth chamber grow plants.

Example 31 Tiller Number Assay with Transgenic Maize PlantsOverexpressing AT-DTP4ac Tiller Production Under Field Conditions

The AT-DTP4 (pCV-DTP4ac) was introduced into a transformable maize linederived from an elite maize inbred line.

Six transgenic events were field tested at 2 locations A (Floweringstress) and B (Well-watered) in 2014. The trials were fieldphysiological frame work. At the location A, mild drought conditionswere imposed during flowering. The “B” location was well-watered. Tillernumber data were collected in all locations, with 4 replicates perlocation. Tiller number per plant was counted for 20 plants in themiddle of plot.

Tiller number (tiller number per plant) for the 6 transgenic events isshown in FIG. 18. Tiller number per plant of transgenic plants wassignificantly greater than construct null (CN).

Example 32 ABA Sensitivity Assay Root and Shoot Growth Assay withAT-DTP4ac Polypeptide in Maize

As described in Examples 5, 7, and 25, overexpressing DTP4 inArabidopsis resulted in increased sensitivity to ABA. To determinewhether transgenic maize plants overexpressing AT-DTP4 (SEQ ID NO:18)were also ABA hypersensitive, a maize ABA assay was performed withtransgenic events and corresponding event nulls of construct pCV-DTP4ac.Maize seeds were germinated in paper towel rolls for 4 days in water,and then either no ABA or 10 μM ABA treatments were applied for 7additional days. Root and shoot growth was measured before and after theABA treatment, and differences were recorded. A positive control eventfrom another construct known to give ABA hypersensitivity was included.Six replications were done, with 5 seeds per germination roll.

Materials and Methods

An experiment with the current protocol was completed in 11 days,starting with germination of seeds in water (0 DAP). After four daysgermination, five seeds of an entry have initial root and shootmeasurements were recorded and were then transferred to an individualgermination roll that has been ascribed with a 10 μM or 0 μM ABAtreatment (0 DAT). Following an additional 7 days in the growth chamber,final root and shoot measurements were recorded for each roll (7 DAT).

Traits were averaged over the five plants in a germination roll. Rootgrowth and shoot growth traits were calculated as the difference of thefinal and initial measurements. Initial measurements were also analyzedto determine if differences were present prior to treatment. Comparisonswere conducted between treatments and entries, on the event andconstruct level using a spatial adjustment. The experimental design wasa multi-time split plot with replications sometimes conducted overseveral days.

Results:

Construct level results from 2 different experiments was done on twodifferent days, results are shown in FIG. 19.

The positive control showed significant decreases in shoot and rootgrowth in the 10 μM ABA treatment, as expected for an ABA hypersensitivecontrol. In contrast, four AT-DTP4ac transgenic events had significantlyincreased root growth, and no events had significantly decreased shootgrowth, suggesting decreased sensitivity to ABA. Thus, overexpressingAT-DTP4 in both Arabidopsis and maize altered ABA sensitivity.

Example 33 Triple Stress Assay with Transgenic Maize PlantsOverexpressing AT-DTP4ac

The triple stress assay was used to test AT-DTP4ac and other AT-DTP4homologs for their ability to confer stress resistance following adrought, light and heat stress combination.

Material and Methods

Maize plants were grown to the V4 stage in a growth chamber underconditions of 27° C. daytime/15° C. nighttime temperatures, 15 hourphotoperiod, 60% relative humidity and 800 μmol m⁻¹ sec⁻¹ lightintensity (Table 11). During this period plants were fertigated tomaintain well-watered conditions. After this 21 day period, initialplant measurements (0 days after treatment, or DAT) were recorded priorto “triple stress”, including volumetric soil water content,hyperspectral imaging, and chlorophyll fluorescence. The triple stresswas initiated by increasing temperatures to 38° C. daytime/27° C.nighttime, increasing the light intensity 1300 μmol m⁻¹ sec⁻¹, and waterwas withheld. Measurements were again collected at 3 and 6 days aftertreatment. At the 6 DAT measurements, plant biomass was destructivelyharvested for fresh and dry weights. Significant differences weredetermined for traits at the event and construct level for 12replicates.

TABLE 11 Experimental Procedure for the Triple Stress Assay Date Event 1week or less prior to Pot filling planting 0 DAP Planting, Start“Normal” Conditions 7 DAP Thinning to 1 plant pot⁻¹ 0 DAT or 20 DAPInitial measurements, Occurs as plants are under “Normal” Conditions 21DAP Growth Chamber Program switches to “Triple Stress” Program 3 DAT or24 DAP Second set of measurements. 6 DAT or 27 DAP Final set ofmeasurements including destructive harvest.

Results: During triple stress, plants with pCV-DTP4ac had greater leafarea compared to null as measured in pixel area with a hyperspectralcamera (FIG. 20). Significant differences were not observed in biomassmeasurements, soil water content or chlorophyll fluorescence parameters.

FIG. 20 shows construct level response of plants with pCV-DTP4ac(UBI:AT-DTP4) for leaf area during triple stress. Significantdifferences are presented at a P<0.1, with black bars indicatingsignificantly positive construct level response, dark grey bars indicatea comparison that is not significantly different. Numbers indicate thepercent difference relative to construct null.

Example 34 Osmotic Stress Assay with Transgenic Maize PlantsOverexpressing AT-DTP4ac

An osmotic stress assay was used to test the ability of DTP4polypeptides to confer osmotic stress resistance in transgenic maizeplants overexpressing DTP4 polypeptides.

These experiments are a variation of the osmotic stress assay describedin Example 25.

Material and Methods:

All experiments were conducted in one Percival growth chamber that ismaintained under completely darkened conditions at 25 degrees C., with arelative humidity of 95%. For each experiment, one construct with allavailable events (transgenics and event nulls) were tested in NuncBioassay Plates (245×245×25 mm, approximately 225 ml volume).

Two treatments were done: control and quad osmotic stress (70%concentration; ψ_(w)=−1.0 MPa)

Each event (transgenic, event null) per treatment contained sixreplicates.

Media Preparation:

-   -   Quad Stress (70%) media:    -   MS Salt—1.1 g/L    -   MES Hydrate-0.3905 g/L    -   PEG 8000—70 g/L    -   Mannitol—15.94 g/L    -   Sorbitol—15.94 g/L    -   NaCl—2.557 g/L    -   Adjust media to 5.70 with 1 M KOH    -   Phytagel—8 g/L    -   Control Media:    -   MS Salt—1.1 g/L    -   MES Hydrate—0.3905 g/L    -   Adjust media to 5.70 with 1 M KOH    -   Phytagel—8 g/L

Results: Seed germination data were collected at 24, 32, 48, 56, 72, and96 hours after plating. The water potentials of the control and quadstress (70% concentration) media were measured via a vapor pressureosmometer at the end of each experiment

Significant inhibition was found in seed germination in response to quadstress, relative to control at 48-96 h. All available events (total ofeight) of PHP51731 were tested twice with reproducible results.AT-DTP4ac transgenic events consistently demonstrated significantlyreduced sensitivity to quad stress, relative to null.

During two experiments, seven of eight transgenic events exhibitedsignificantly reduced germination sensitivity to quad stress, relativeto comparable nulls.

Results are shown in Table 12 and FIG. 21.

TABLE 12 Osmotic Stress Assay With AT-DTP4 Overexpressing Maize PlantsConstruct Control Quad Stress pCV-DTP4ac Neutral Positive (p < 0.05)

Example 35 Tall Clear Tube Root Growth Assay with Transgenic MaizePlants Overexpressing AT-DTP4ac to Evaluate Root and Shoot Development

This assay was developed and used to evaluate root growth developmentalplasticity in transgenic maize plants overexpressing DTP4 polypeptidesin response to well-watered and soil drying conditions.

Material and Methods:

The experiments were performed in greenhouse. Maize seeds were imbibedon germination paper that was pre-soaked in water for a 48 h period.Uniform maize seedlings (with root lengths between 10-22 mm) weretransplanted into clear acrylic tubes (1.5 meters in length,approximately 38 L volume) containing a 3:1 Dynamix to sand media. Thesoil media was supplemented with Scott's Osmocote Plus (15-9-12) toprovide a slow release of nutrients throughout the course of eachexperiment. For each experiment, one construct with two selected events(transgenic and event null) were tested. Two treatments were done: wellwatered and drought. The drought cycle was induced between V3-V4 growthstages, for three weeks. Each event (transgenic, event null) pertreatment contained 6 replicates.

Measurements were done to monitor lateral growth development with depthand time, a total of 40 root windows were permanently installed by acustom fabrication vendor, according to design specifications. Todelineate the differing depths, each root window has been systematicallyassigned a number designation. Lateral root growth is monitored on aweekly basis following water withholding by taking a series ofphotographs of each root window at the different depth increments with adigital camera with an attached polarizing filter. To ensure thatstandardized photographs were taken, the camera is installed on acustomized designed and fabricated acrylic jig. All images were sent forautomated quantitative analysis.

Soil water content measurements: The apparent dielectric constant of theuppermost 100 cm of soil was quantified bi-weekly using a soil moistureprobe in all plants during the drought period to better interpret aswell as compare the timing and pattern of root development both withinas well as between genotypes. Plant growth quantification: plant heightand leaf number data were collected bi-weekly, during the droughtperiod. The harvest measurements done were for shoot fresh weight, shootdry weight, total leaf area, primary root length; data were collectedfor all plants.

TABLE 13 Tall Clear Tube Root Assay With AT-DTP4 Overexpressing MaizePlants Construct Promoter Events Treatment Major Difference pCV- ZM-UBIDTP4-L17 (TG, WW, Soil TG positive DTP4ac event null) drying producedmore tillers, under WW conditions, relative to null DTP4-L16 (TG, WW,Soil TG positive event null) drying produced more tillers, under WWconditions, relative to null

Example 36 Expression of AT-DTP4 Fusion Protein in E. coli ProteinPurification and Esterase Activity Assays

The pET28a expression vector was used to express AT-DTP4 fusion proteincontaining 20 additional N-terminal amino acids, including a 6 histidinetag. The amino acid sequence of the fusion protein is presented as SEQID NO:629. E. coli cultures were grown at 37° C. in 2×YT media to anOD_(600nm) of 0.6. Transgene expression was then induced with 0.5 mMIPTG and the culture was grown an additional 20 hours at 20° C. Thefusion protein was purified from E. coli extracts using cobalt affinitychromatography, and a high degree of purity was achieved. Aliquots ofthe purified protein were stored frozen at −80° C. in 10% glycerol.Aliquots were then thawed and dialyzed against 50 mM Tris-HCl pH 8,prior to performing esterase activity assays with p-nitrophenyl acetateas substrate.

Esterase activity with this substrate was monitored by observing anincrease in absorbance at a wavelength of 405 nm, because thep-nitrophenol product absorbs at 405 nm. The activity assays were donewith 1 μg of protein in 50 mM Tris-HCl, pH 8, with an assay volume of200 μl, using 96 well flat bottom microtiter plates. Control reactionswithout enzyme were done and rates were subtracted from the plus enzymereaction rates to correct for autohydrolysis of substrate. The purifiedAT-DTP4 protein had obvious esterase activity with p-nitrophenyl acetateas substrate (FIG. 23). Dialyzed protein was quantitated by absorbanceat 280 nm, using a value of 1 OD (280 nm)=0.92 mg/ml.

Example 37 Traits Observed in Field Plots in Transgenic Maize PlantsOverexpressing AT-DTP4 Polypeptide

Field plots were observed in well watered conditions with transgenicmaize plants transformed with pCV-DTP4ac. A randomized complete blockdesign was used with 2 row plots and 4 field replications. Fiveconsecutive evenly spaced plants in each row were tagged forobservation, for a total of 10 plants per plot. In some plots, fewerthan 10 plants were used for observations. For one trait, tiller numberat V12, all the plants of a plot were used, except for the end plant oneach side of each row. For another trait, stalk diameter, only 3 eventswere measured. Descriptions of the traits measured, a summary of theresults are presented in Table 14, and detailed results are presented inTable 15. At the construct level, small but statistically significantdifferences from nulls were observed for several traits, includingdecreases in plant height at V12, leaf number at V9, and growth ratefrom V9 to V12. Increased tiller number was observed at V12. Pollen shedwas about half a day later, and because silks emerged before pollen shedin these well watered conditions, the ASI was negative and larger due tothe delayed shed.

TABLE 14 Trait Description and Result Summary in Field Plots Significantdifference from null (construct Trait Description level) PLTHT.V9 Plantheight (cm) at V9, to top leaf collar No LFN.V9 Leaf number with visiblecollar at V9 Yes; decreased TILN.V12 Tiller number at V12 Yes; increasedPLTHT.V12 Plant height (cm) at V12, to top leaf Yes; collar decreasedLFN.V12 Leaf number with visible collar at V12 No PLTHT.V17 Plant height(cm) at V17 to V18, to top No leaf collar GR.V9V12 Growth rate (cm/day)from June 23 to Yes; July 3 (V9 to V12). decreased GR.V12V17 Growth rate(cm/day) from June 23 to No July 3 (V9 to V12). SHED First pollen shedfor 50% of plants in Yes; later plot, days after planting SILK Firstsilk emergence for 50% of plants in No plot, days after planting ASIAnthesis to silk interval (silk date - shed Yes; larger date) PLTHT.R3Plant height (cm), final, to bottom of No tassel EARHT Ear height (cm)No LFN.R3 Leaf number, final No EARLP Ear leaf position No STKD Stalkdiameter (cm), perpendicular to No groove, mid internode below earSTAGRN.ER4 Stay green, lowest leaf that is >50% No green, ear leafposition = 0, Very early R4. STAGRN.R4 Stay green, lowest leaf thatis >50% No green, ear leaf position = 0. R4.

TABLE 15 Traits Observed In Field Plots significantly different fromnull (** p value <0.05; mean Difference * p Value Trait Event value fromnull p Value <0.1) PLTHT.V9 DTP4-L10 50.07 −1.51 0.182 PLTHT.V9 DTP4-L1150.41 −1.17 0.308 PLTHT.V9 DTP4-L12 50.69 −0.90 0.449 PLTHT.V9 DTP4-L1351.69 0.10 0.928 PLTHT.V9 DTP4-L14 50.27 −1.32 0.234 PLTHT.V9 DTP4-L1550.91 −0.68 0.567 PLTHT.V9 DTP4-L16 51.94 0.36 0.747 PLTHT.V9 DTP4-L1750.18 −1.40 0.226 PLTHT.V9 Construct 50.77 −0.82 0.402 PLTHT.V9 null51.59 0.00 LFN.V9 DTP4-L10 8.89 −0.11 0.012 ** LFN.V9 DTP4-L11 8.82−0.19 0.000 ** LFN.V9 DTP4-L12 8.94 −0.06 0.163 LFN.V9 DTP4-L13 8.97−0.04 0.393 LFN.V9 DTP4-L14 8.90 −0.10 0.016 ** LFN.V9 DTP4-L15 8.92−0.08 0.083 * LFN.V9 DTP4-L16 8.97 −0.04 0.409 LFN.V9 DTP4-L17 8.88−0.12 0.007 ** LFN.V9 Construct 8.91 −0.09 0.018 ** LFN.V9 null 9.000.00 TILN.V12 DTP4-L10 0.07 0.06 0.035 ** TILN.V12 DTP4-L11 0.06 0.060.063 * TILN.V12 DTP4-L12 0.07 0.06 0.051 * TILN.V12 DTP4-L13 0.06 0.050.115 TILN.V12 DTP4-L14 0.09 0.08 0.013 ** TILN.V12 DTP4-L15 0.07 0.060.039 ** TILN.V12 DTP4-L16 0.09 0.08 0.009 ** TILN.V12 DTP4-L17 0.140.13 0.000 ** TILN.V12 Construct 0.08 0.07 0.006 ** TILN.V12 null 0.010.00 PLTHT.V12 DTP4-L10 98.99 −3.28 0.033 ** PLTHT.V12 DTP4-L11 99.11−3.16 0.042 ** PLTHT.V12 DTP4-L12 98.68 −3.59 0.024 ** PLTHT.V12DTP4-L13 99.60 −2.67 0.080 * PLTHT.V12 DTP4-L14 98.72 −3.55 0.020 **PLTHT.V12 DTP4-L15 99.19 −3.08 0.050 * PLTHT.V12 DTP4-L16 99.86 −2.410.112 PLTHT.V12 DTP4-L17 99.01 −3.25 0.036 ** PLTHT.V12 Construct 99.14−3.12 0.026 ** PLTHT.V12 null 102.27 0.00 LFN.V12 DTP4-L10 11.75 −0.130.162 LFN.V12 DTP4-L11 11.73 −0.15 0.111 LFN.V12 DTP4-L12 11.73 −0.140.120 LFN.V12 DTP4-L13 11.77 −0.11 0.232 LFN.V12 DTP4-L14 11.76 −0.110.214 LFN.V12 DTP4-L15 11.74 −0.13 0.147 LFN.V12 DTP4-L16 11.78 −0.100.268 LFN.V12 DTP4-L17 11.76 −0.12 0.198 LFN.V12 Construct 11.75 −0.120.145 LFN.V12 null 11.88 0.00 PLTHT.V17 DTP4-L10 195.38 −1.80 0.400PLTHT.V17 DTP4-L11 195.26 −1.92 0.376 PLTHT.V17 DTP4-L12 194.76 −2.420.275 PLTHT.V17 DTP4-L13 196.13 −1.05 0.621 PLTHT.V17 DTP4-L14 194.78−2.40 0.257 PLTHT.V17 DTP4-L15 195.97 −1.21 0.582 PLTHT.V17 DTP4-L16196.36 −0.82 0.699 PLTHT.V17 DTP4-L17 194.82 −2.36 0.278 PLTHT.V17Construct 195.43 −1.75 0.367 PLTHT.V17 null 197.18 0.00 GR.V9V12DTP4-L10 4.84 −0.22 0.006 ** GR.V9V12 DTP4-L11 4.84 −0.22 0.006 **GR.V9V12 DTP4-L12 4.84 −0.22 0.006 ** GR.V9V12 DTP4-L13 4.84 −0.22 0.006** GR.V9V12 DTP4-L14 4.84 −0.22 0.006 ** GR.V9V12 DTP4-L15 4.84 −0.220.006 ** GR.V9V12 DTP4-L16 4.84 −0.22 0.006 ** GR.V9V12 DTP4-L17 4.84−0.22 0.006 ** GR.V9V12 Construct 4.84 −0.22 0.006 ** GR.V9V12 null 5.060.00 GR.V12V17 DTP4-L10 8.76 0.12 0.170 GR.V12V17 DTP4-L11 8.75 0.120.183 GR.V12V17 DTP4-L12 8.75 0.12 0.188 GR.V12V17 DTP4-L13 8.76 0.120.167 GR.V12V17 DTP4-L14 8.75 0.12 0.184 GR.V12V17 DTP4-L15 8.76 0.130.155 GR.V12V17 DTP4-L16 8.76 0.12 0.170 GR.V12V17 DTP4-L17 8.75 0.110.202 GR.V12V17 Construct 8.76 0.12 0.168 GR.V12V17 null 8.64 0.00PLTHT.R3 DTP4-L10 264.02 −0.66 0.703 PLTHT.R3 DTP4-L11 264.02 −0.660.703 PLTHT.R3 DTP4-L12 264.02 −0.66 0.703 PLTHT.R3 DTP4-L13 264.02−0.66 0.703 PLTHT.R3 DTP4-L14 264.02 −0.66 0.703 PLTHT.R3 DTP4-L15264.02 −0.66 0.703 PLTHT.R3 DTP4-L16 264.02 −0.66 0.703 PLTHT.R3DTP4-L17 264.02 −0.66 0.703 PLTHT.R3 Construct 264.02 −0.66 0.703PLTHT.R3 null 264.68 0.00 EARHT DTP4-L10 105.34 1.98 0.243 EARHTDTP4-L11 105.34 1.98 0.243 EARHT DTP4-L12 105.34 1.98 0.243 EARHTDTP4-L13 105.34 1.98 0.243 EARHT DTP4-L14 105.34 1.98 0.243 EARHTDTP4-L15 105.34 1.98 0.243 EARHT DTP4-L16 105.34 1.98 0.243 EARHTDTP4-L17 105.34 1.98 0.243 EARHT Construct 105.34 1.98 0.243 EARHT null103.36 0.00 LFN.R3 DTP4-L10 18.63 −0.24 0.024 ** LFN.R3 DTP4-L11 18.67−0.20 0.060 LFN.R3 DTP4-L12 18.79 −0.08 0.460 LFN.R3 DTP4-L13 18.84−0.03 0.793 LFN.R3 DTP4-L14 18.83 −0.04 0.655 LFN.R3 DTP4-L15 18.85−0.02 0.870 LFN.R3 DTP4-L16 18.83 −0.04 0.722 LFN.R3 DTP4-L17 18.84−0.03 0.789 LFN.R3 Construct 18.79 −0.08 0.344 LFN.R3 null 18.87 0.00EARLP DTP4-L10 11.89 −0.01 0.927 EARLP DTP4-L11 11.92 0.02 0.793 EARLPDTP4-L12 11.95 0.06 0.527 EARLP DTP4-L13 11.95 0.06 0.521 EARLP DTP4-L1412.01 0.12 0.192 EARLP DTP4-L15 11.93 0.04 0.672 EARLP DTP4-L16 11.960.07 0.455 EARLP DTP4-L17 11.95 0.05 0.564 EARLP Construct 11.95 0.050.523 EARLP null 11.89 0.00 Shed DTP4-L10 70.37 0.40 0.215 Shed DTP4-L1170.37 0.40 0.215 Shed DTP4-L12 70.46 0.49 0.126 Shed DTP4-L13 70.27 0.300.342 Shed DTP4-L14 70.46 0.49 0.126 Shed DTP4-L15 70.65 0.68 0.037 **Shed DTP4-L16 70.27 0.30 0.342 Shed DTP4-L17 70.65 0.68 0.037 ** ShedConstruct 70.44 0.47 0.095 * Shed null 69.97 0.00 Silk DTP4-L10 69.550.05 0.877 Silk DTP4-L11 69.58 0.08 0.799 Silk DTP4-L12 69.61 0.11 0.723Silk DTP4-L13 69.55 0.05 0.877 Silk DTP4-L14 69.61 0.11 0.723 SilkDTP4-L15 69.61 0.11 0.723 Silk DTP4-L16 69.55 0.05 0.877 Silk DTP4-L1769.67 0.17 0.579 Silk Construct 69.59 0.09 0.757 Silk null 69.51 0.00ASI DTP4-L10 −0.84 −0.42 0.063 * ASI DTP4-L11 −0.84 −0.42 0.063 * ASIDTP4-L12 −0.84 −0.42 0.063 * ASI DTP4-L13 −0.84 −0.42 0.063 * ASIDTP4-L14 −0.84 −0.42 0.063 * ASI DTP4-L15 −0.84 −0.42 0.063 * ASIDTP4-L16 −0.84 −0.42 0.063 * ASI DTP4-L17 −0.84 −0.42 0.063 * ASIConstruct −0.84 −0.42 0.063 * ASI null −0.43 0.00 STKD DTP4-L13 17.18−0.13 0.275 STKD DTP4-L16 17.18 −0.13 0.275 STKD DTP4-L17 17.18 −0.130.275 STKD Construct 17.18 −0.13 0.275 STKD null 17.31 0.00 STAGRN.ER4DTP4-L10 −3.41 −0.08 0.476 STAGRN.ER4 DTP4-L11 −3.41 −0.08 0.476STAGRN.ER4 DTP4-L12 −3.41 −0.08 0.476 STAGRN.ER4 DTP4-L13 −3.41 −0.080.476 STAGRN.ER4 DTP4-L14 −3.41 −0.08 0.476 STAGRN.ER4 DTP4-L15 −3.41−0.08 0.476 STAGRN.ER4 DTP4-L16 −3.41 −0.08 0.476 STAGRN.ER4 DTP4-L17−3.41 −0.08 0.476 STAGRN.ER4 Construct −3.41 −0.08 0.476 STAGRN.ER4 null−3.32 0.00 STAGRN.R4 DTP4-L10 −2.26 −0.23 0.129 STAGRN.R4 DTP4-L11 −2.15−0.12 0.447 STAGRN.R4 DTP4-L12 −2.20 −0.17 0.296 STAGRN.R4 DTP4-L13−2.10 −0.07 0.664 STAGRN.R4 DTP4-L14 −2.14 −0.11 0.476 STAGRN.R4DTP4-L15 −2.25 −0.22 0.165 STAGRN.R4 DTP4-L16 −2.04 −0.01 0.950STAGRN.R4 DTP4-L17 −2.27 −0.24 0.124 STAGRN.R4 Construct −2.18 −0.150.272 STAGRN.R4 null −2.03 0.00

Example 38 Traits Observed in Field Pots

In addition to the field plots described in Example 37, a field potstudy was also performed at a well-watered location. Growing maizeplants in pots allowed the option of imposing drought stress in awell-watered location by irrigating less, because plants in potsreceived more water from irrigation than from rainfall, due to the smallneck size of the pots and the fact that water drained quickly from pots.The pots were 10 liter volume, 7.75″×18″ square treepots. A split plotdesign was used, with treatment being the whole plot, event the splitplot, and transgenic event and event null the split plot. So throughoutthe experiment, each event was adjacent to its corresponding event null.There were six pots per replication, comprising three transgenic eventsand the three corresponding event nulls. 30 replications in the wellwatered treatment and 30 replications in the drought stressed treatmentwere done. In each treatment, 15 of the 30 reps were harvested at R1,and the other 15 reps were harvested at R6. Descriptions of the traitsmeasured, and a summary of the results for the pot study are presentedin Table 16, and results are presented in Table 17. At the constructlevel in the well watered treatment, significant differences from nullswere observed for the following traits: increased tiller number at V4and V6, reduced plant height at V10, V13, V16, and R1, reduced leafnumber at V10, decreased growth rate from V6 to V10, decreasedflavonols, decreased water use efficiency, decreased dry weight of themain shoot at R1, increased dry weight of tillers at R1, delayed shedand silk time, and increased vegetative dry weight at R6. At theconstruct level in the drought stressed treatment, significantdifferences from nulls were observed for the following traits: increasedtillers at V4 and V6, decreased plant height at V6, V10, and V13,decreased leaf number at V10, V13, and at maturity, decreased flavonols,decreased dry weight of the main shoot at R1, increased dry weight oftillers and ear at R1, earlier silking time, decreased ASI, decreasedyellow leaves (increased stay green) at 3 dates, decreased vegetativedry weight at R6, and increased dry weight of kernels (yield), ear,kernel number, and harvest index at R6. A summary is given in Table 16,and the numbers for different events are given in Table 17.

Significance of many of these traits in determining plant health, yieldand biomass are well known in the art. For example, chlorophyll andflavonol measurement using Dualex instrument, measurement of othertraits such as harvest index, water use efficiency, plant height, dryweight, kernel weight etc is well known in the art (Cerovic et alPhysiologia Plantarum 146: 251-260. 2012; Sinclair, T. R.; Crop Sci.38:638-643(1998), Edmeades et al (1999) Crop Sci. 39:1306-1315, Andradeet al Crop Sci. 42:1173-1179 (2002), Berke et al (1995) Crop Sci.39:1542-1549, Garwood et al Crop Science, Vol. 10, January-February1970).

TABLE 16 Trait Descriptions for Field Pot Study Significant Significantdifference from difference from null in ww null in drought Trait Traitdescription (construct level) (construct level) TILN.V4 Tiller number atV4 Yes; increased Yes; increased PLTHT.V6 Plant height (cm) at V6 NoYes; decreased LFN.V6 Leaf number with visible collar No No at V6TILN.V6 Tiller number at V6 Yes; increased Yes; increased PLTHT.V10Plant height (cm) at V10 Yes; decreased Yes; decreased LFN.V10 Leafnumber with visible collar Yes; decreased Yes; decreased at V10PLTHT.V13 Plant height (cm) at V13 Yes; decreased Yes; decreased LFN.V13Leaf number with visible collar Yes; decreased No at V13 PLTHT.V16 Plantheight (cm), about V16 Yes; decreased No and V15 for WW and DRT.DUALEX.CHL Chlorophyll by Dualex No No instrument, near middle of 11thleaf. DUALEX.FLV Flavonols by Dualex Yes; decreased Yes; decreasedinstrument, near middle of 11th leaf. DUALEX.NBI Nitrogen band index byDualex No No instrument, near middle of 11th leaf. RWC Relative watercontent (%), ND No 10th leaf, R1 DRT pots WU Water use (initial potweight − No ND 24 hr pot weight, WW R1) WUE Water use/total biomass Yes;decreased ND PLTHT.R1 Plant height (cm) at R1 Yes; decreased No PLTHT.R6Plant height (cm) at R6 ND ND EARHT Ear height (cm), final No No LFNLeaf number, final No Yes; decreased EARLP Position of ear leaf (donefor No Yes; negative R6 plants only) DWMAIN.R1 Dry weight of main plantat R1 Yes; decreased Yes; decreased DWTIL.R1 Dry weight of tillers at R1Yes; increased Yes; increased DWVEG.R1 Dry weight of mainplant and Yes;decreased No tillers, minus ear, R1 DWEAR.R1 Dry weight of primary earat Yes; decreased Yes, increased R1 DWTOT.R1 Dry weight of total plantat R1 Yes; decreased No SHED First pollen shed Yes; delayed No SILKFirst silk emergence Yes, delayed Yes; earlier ASI Silk date − shed dateNo Yes; decreased GR.V6V10 Growth rate (cm/day) from Yes; decreased Yes;decreased June 18 to July 1 (V6 to V10) GR.V10V13 Growth rate (cm/day)from No No July 1 to July 9 (V10 to V13) YL.date 1 Number of leaves >50%ND Yes; decreased yellow on date 1 YL.date 2 Number of leaves >50% NDYes; decreased yellow on date 2 YL.date 3 Number of leaves >50% ND Yes;decreased yellow on date 3 DWVEG.R6 Dry weight (g) at R6 of all Yes;increased Yes; decreased plant parts except for ear ROW Rows of kernels(rows are the No No long way) DWK Dry weight of kernels (g). No Yes;increased (yield) DWCOB Dry weight of cob (g) (by math, No No DWEAR −DWK) DWEAR Dry weight of ear (g) No Yes; increased KN Kernel number NoYes; increased X100KW 100 kernel dry weight (g) No No DWTOT Dry weightof total plant (by No No math, DWVEG.R6 + EARW) HI Harvest index,DWK/DWTOT No Yes; increased at R6 ND: “not determined”

TABLE 17 Traits Observed in Field Pots. Event Event or null orsignificantly TREAT- Event or construct construct Difference differentMENT TRAIT construct mean null mean from null p Value from null WWTILN.V4 DTP4-L13 1.26 0.97 0.29 0.10061240 WW TILN.V4 DTP4-L16 1.37 0.970.40 0.02068587 ** WW TILN.V4 DTP4-L17 1.36 0.94 0.42 0.01560281 ** WWTILN.V4 Construct 1.33 0.96 0.37 0.00027952 ** WW PLTHT.V6 DTP4-L1322.37 22.21 0.16 0.57160892 WW PLTHT.V6 DTP4-L16 21.98 22.05 −0.060.82730554 WW PLTHT.V6 DTP4-L17 21.50 22.13 −0.83 0.02710131 ** WWPLTHT.V6 Construct 21.95 22.13 −0.18 0.28166786 WW LFN.V6 DTP4-L13 5.935.83 0.10 0.24382458 WW LFN.V6 DTP4-L16 5.83 5.83 0.00 1.00000000 WWLFN.V6 DTP4-L17 5.86 5.93 −0.07 0.42198554 WW LFN.V6 Construct 5.88 5.870.01 0.83305644 WW TILN.V6 DTP4-L13 2.69 2.37 0.33 0.01376576 ** WWTILN.V6 DTP4-L16 2.67 2.27 0.40 0.00242878 ** WW TILN.V6 DTP4-L17 2.662.40 0.26 0.05289162 * WW TILN.V6 Construct 2.67 2.34 0.33 0.00002468 **WW PLTHT.V10 DTP4-L13 88.91 90.98 −2.07 0.05629611 * WW PLTHT.V10DTP4-L16 87.84 91.09 −3.25 0.00269623 ** WW PLTHT.V10 DTP4-L17 82.8589.11 −6.46 0.00000001 ** WW PLTHT.V10 Construct 86.47 90.39 −3.930.00000000 ** WW LFN.V10 DTP4-L13 10.04 10.07 −0.03 0.61109281 WWLFN.V10 DTP4-L16 9.93 9.97 −0.03 0.58640533 WW LFN.V10 DTP4-L17 9.8310.00 −0.17 0.00599107 ** WW LFN.V10 Construct 9.93 10.01 −0.080.02779416 ** WW PLTHT.V13 DTP4-L13 137.88 139.92 −2.05 0.10694161 WWPLTHT.V13 DTP4-L16 138.94 139.32 −2.37 0.05908537 * WW PLTHT.V13DTP4-L17 130.10 137.89 −7.79 0.00000001 ** WW PLTHT.V13 Construct 134.97139.04 −4.07 0.00000012 ** WW LFN.V13 DTP4-L13 13.04 13.07 −0.030.66799636 WW LFN.V13 DTP4-L16 12.97 13.00 −0.03 0.64259662 WW LFN.V13DTP4-L17 12.90 13.03 −0.14 0.06236167 * WW LFN.V13 Construct 12.97 13.03−0.07 0.11105689 WW PLTHT.V16 DTP4-L13 190.40 193.60 −3.20 0.02670512 **WW PLTHT.V16 DTP4-L16 187.88 192.08 −4.20 0.00336881 ** WW PLTHT.V16DTP4-L17 179.91 188.77 −8.86 0.00000001 ** WW PLTHT.V16 Construct 186.06191.48 −5.42 0.00000000 ** WW DUALEX.CHL DTP4-L13 45.09 44.69 0.400.78788150 WW DUALEX.CHL DTP4-L16 43.87 44.62 −0.75 0.62014378 WWDUALEX.CHL DTP4-L17 43.18 44.79 −1.63 0.27233009 WW DUALEX.CHL Construct44.04 44.70 −0.66 0.44885445 WW DUALEX.FLV DTP4-L13 0.83 0.88 −0.060.34590013 WW DUALEX.FLV DTP4-L16 0.84 0.88 −0.03 0.58952091 WWDUALEX.FLV DTP4-L17 0.80 0.89 −0.09 0.11031364 WW DUALEX.FLV Construct0.82 0.88 −0.06 0.08113570 * WW DUALEX.NBI DTP4-L13 59.53 54.80 4.730.23817731 WW DUALEX.NBI DTP4-L16 55.40 53.47 1.93 0.62848327 WWDUALEX.NBI DTP4-L17 57.27 53.35 3.92 0.33121059 WW DUALEX.NBI Construct57.40 53.87 3.53 0.13539637 WW WU DTP4-L13 1168.57 1104.31 64.260.06497045 * WW WU DTP4-L16 1127.24 1009.67 117.57 0.00144349 ** WW WUDTP4-L17 1015.46 1112.83 −97.36 0.00628317 ** WW WU Construct 1103.761075.60 28.16 0.17655244 WW WUE DTP4-L13 0.13 0.14 −0.01 0.04438817 **WW WUE DTP4-L16 0.13 0.14 −0.01 0.00774256 ** WW WUE DTP4-L17 0.13 0.13−0.01 0.13121996 WW WUE Construct 0.13 0.14 −0.01 0.00079553 ** WWPLTHT.R1 DTP4-L13 261.68 259.12 2.56 0.24461804 WW PLTHT.R1 DTP4-L16256.35 252.12 4.22 0.03844392 ** WW PLTHT.R1 DTP4-L17 246.04 260.39−14.35 0.00000000 ** WW PLTHT.R1 Construct 254.69 257.21 −2.520.01432462 ** WW EARHT DTP4-L13 104.02 108.16 −4.14 0.18688931 WW EARHTDTP4-L16 113.19 109.85 3.34 0.26579653 WW EARHT DTP4-L17 108.27 109.87−1.60 0.58734218 WW EARHT Construct 108.50 109.30 −0.80 0.63293108 WWLFN DTP4-L13 18.90 18.87 0.03 0.80990694 WW LFN DTP4-L16 18.93 18.870.06 0.60450049 WW LFN DTP4-L17 18.86 18.73 0.13 0.30082730 WW LFNConstruct 18.90 18.82 0.07 0.30080168 WW EARLP DTP4-L13 11.01 11.34−0.33 0.02957145 ** WW EARLP DTP4-L16 11.14 11.27 −0.13 0.37839219 WWEARLP DTP4-L17 11.51 11.47 0.03 0.82043803 WW EARLP Construct 11.2211.36 −0.14 0.10366448 WW DWMAIN.R1 DTP4-L13 141.28 154.43 −13.140.00329335 ** WW DWMAIN.R1 DTP4-L16 135.89 141.91 −6.02 0.14381188 WWDWMAIN.R1 DTP4-L17 126.35 145.84 −19.49 0.00000722 ** WW DWMAIN.R1Construct 134.51 147.39 −12.89 0.00000011 ** WW DWTIL.R1 DTP4-L13 10.706.90 3.80 0.01819417 ** WW DWTIL.R1 DTP4-L16 11.45 5.30 6.140.00035140 * WW DWTIL.R1 DTP4-L17 10.53 8.63 1.91 0.29347209 WW DWTIL.R1Construct 10.89 6.94 3.95 0.00000977 ** WW DWVEG.R1 DTP4-L13 154.58159.77 −5.19 0.32753594 WW DWVEG.R1 DTP4-L16 149.77 145.31 4.470.39342235 WW DWVEG.R1 DTP4-L17 137.68 154.33 −16.65 0.00206288 ** WWDWVEG.R1 Construct 147.34 153.14 −5.79 0.06184574 * WW DWEAR.R1 DTP4-L132.40 2.27 0.13 0.46947926 WW DWEAR.R1 DTP4-L16 2.23 2.01 0.23 0.19670390WW DWEAR.R1 DTP4-L17 1.27 2.18 −0.92 0.00000063 ** WW DWEAR.R1 Construct1.97 2.15 −0.18 0.07229087 * WW DWTOT.R1 DTP4-L13 158.68 162.14 −5.460.30802330 WW DWTOT.R1 DTP4-L16 151.81 147.25 4.56 0.38844940 WWDWTOT.R1 DTP4-L17 139.30 156.50 −17.20 0.00164783 ** WW DWTOT.R1Construct 149.28 155.30 −6.03 0.05436711 ** WW SHED DTP4-L13 61.47 61.440.03 0.83820281 WW SHED DTP4-L16 81.59 61.46 0.14 0.36890286 WW SHEDDTP4-L17 61.99 61.45 0.54 0.00066393 ** WW SHED Construct 61.69 61.450.24 0.00828503 ** WW SILK DTP4-L13 62.48 62.42 0.06 0.74458746 WW SILKDTP4-L16 62.73 62.48 0.26 0.12897308 WW SILK DTP4-L17 62.90 62.50 0.400.02421494 ** WW SILK Construct 62.70 62.47 0.24 0.01767604 ** WW ASIDTP4-L13 1.00 0.97 0.03 0.88029003 WW ASI DTP4-L16 1.13 1.00 0.130.52025914 WW ASI DTP4-L17 0.89 1.04 −0.15 0.48299805 WW ASI Construct1.01 1.00 0.00 0.96659995 WW GR.V6V10 DTP4-L13 5.12 5.29 −0.160.02138426 ** WW GR.V6V10 DTP4-L16 5.05 5.30 −0.25 0.00051092 ** WWGR.V6V10 DTP4-L17 4.72 5.16 −0.44 0.00000000 ** WW GR.V6V10 Construct4.96 5.25 −0.28 0.00000000 ** WW GR.V10V13 DTP4-L13 6.05 6.06 0.000.97370623 WW GR.V10V13 DTP4-L16 6.05 5.99 0.05 0.57289713 WW GR.V10V13DTP4-L17 5.90 5.99 −0.09 0.34364716 WW GR.V10V13 Construct 6.00 6.01−0.01 0.80674055 WW STKD DTP4-L13 19.66 19.57 0.09 0.81915673 WW STKDDTP4-L16 19.41 19.14 0.26 0.50007008 WW STKD DTP4-L17 18.47 19.44 −0.970.01514156 ** WW STKD Construct 19.18 19.38 −0.21 0.36986927 WW DWVEG.R6DTP4-L13 185.19 177.14 8.05 0.472471015 WW DWVEG.R6 DTP4-L16 186.95160.32 26.63 0.020291776 ** WW DWVEG.R6 DTP4-L17 179.65 173.34 6.300.559822469 WW DWVEG.R6 Construct 183.93 170.27 13.66 0.035936981 ** WWROW DTP4-L13 15.59 15.91 −0.32 0.479182267 WW ROW DTP4-L16 15.46 15.230.23 0.617709691 WW ROW DTP4-L17 15.67 15.46 0.21 0.637947442 WW ROWConstruct 15.57 15.53 0.04 0.874855288 WW DWK DTP4-L13 191.37 198.81−7.44 0.438648208 WW DWK DTP4-L16 185.09 182.01 3.08 0.752787745 WW DWKDTP4-L17 201.76 190.13 11.63 0.234351744 WW DWK Construct 192.74 190.322.42 0.666015774 WW DWCOB DTP4-L13 29.01 30.61 −1.60 0.30686635 WW DWCOBDTP4-L16 28.21 27.05 1.16 0.46548495 WW DWCOB DTP4-L17 29.98 29.34 0.640.688236714 WW DWCOB Construct 29.07 29.00 0.07 0.940788565 WW DWEARDTP4-L13 220.38 229.44 −9.06 0.408924123 WW DWEAR DTP4-L16 213.27 209.024.25 0.703637408 WW DWEAR DTP4-L17 231.74 219.47 12.28 0.271297248 WWDWEAR Construct 221.80 219.31 2.49 0.697630835 WW KN DTP4-L13 622.83672.22 −49.39 0.094181834 WW KN DTP4-L16 596.25 605.66 −9.41 0.75222573WW KN DTP4-L17 650.05 614.89 35.16 0.238313504 WW KN Construct 623.04630.92 −7.88 0.645325941 WW X100KW DTP4-L13 30.64 29.59 1.05 0.253513862WW X100KW DTP4-L16 31.21 30.16 1.05 0.262791041 WW X100KW DTP4-L17 31.0030.80 0.20 0.833547395 WW X100KW Construct 30.95 30.18 0.77 0.15555604WW DWTOT DTP4-L13 411.37 413.61 −2.24 0.916063317 WW DWTOT DTP4-L16404.92 374.02 30.90 0.15154254 WW DWTOT DTP4-L17 420.11 393.88 26.230.203301266 WW DWTOT Construct 412.14 393.84 18.30 0.136197041 WW HIDTP4-L13 0.48 0.49 −0.02 0.257690608 WW HI DTP4-L16 0.47 0.50 −0.030.035508997 ** WW HI DTP4-L17 0.50 0.48 0.02 0.200998462 WW HI Construct0.48 0.49 −0.01 0.236486772 DRT TILN.V4 DTP4-L13 1.18 0.81 0.370.03737189 ** DRT TILN.V4 DTP4-L16 1.34 0.61 0.73 0.00004332 ** DRTTILN.V4 DTP4-L17 1.28 0.74 0.53 0.00263586 ** DRT TILN.V4 Construct 1.270.72 0.54 0.00000022 ** DRT PLTHT.V6 DTP4-L13 22.11 22.20 −0.090.71441900 DRT PLTHT.V6 DTP4-L16 21.77 21.84 −0.07 0.78976699 DRTPLTHT.V6 DTP4-L17 21.57 22.18 −0.61 0.01802070 ** DRT PLTHT.V6 Construct21.81 22.07 −0.26 0.08476641 ** DRT LFN.V6 DTP4-L13 5.98 5.95 0.030.45412771 DRT LFN.V6 DTP4-L16 5.95 5.95 0.00 1.00000000 DRT LFN.V6DTP4-L17 5.88 5.98 −0.10 0.02565660 ** DRT LFN.V6 Construct 5.94 5.96−0.02 0.38752173 DRT TILN.V6 DTP4-L13 2.60 2.17 0.43 0.00206163 ** DRTTILN.V6 DTP4-L16 2.77 2.10 0.67 0.00000321 ** DRT TILN.V6 DTP4-L17 2.802.37 0.43 0.00206163 ** DRT TILN.V6 Construct 2.72 2.21 0.51 0.00000000** DRT PLTHT.V10 DTP4-L13 92.20 93.42 −1.21 0.25004790 DRT PLTHT.V10DTP4-L16 91.37 92.32 −0.95 0.37703904 DRT PLTHT.V10 DTP4-L17 88.49 92.79−4.30 0.00007419 ** DRT PLTHT.V10 Construct 90.69 92.84 −2.15 0.00066349** DRT LFN.V10 DTP4-L13 10.00 10.07 −0.07 0.30588796 DRT LFN.V10DTP4-L16 10.07 10.03 0.03 0.60828418 DRT LFN.V10 DTP4-L17 9.93 10.10−0.17 0.01109122 ** DRT LFN.V10 Construct 10.00 10.07 −0.07 0.07704135DRT PLTHT.V13 DTP4-L13 135.66 138.16 −2.50 0.05873323 * DRT PLTHT.V13DTP4-L16 135.10 134.55 0.56 0.67020840 DRT PLTHT.V13 DTP4-L17 134.27137.87 −3.60 0.00667808 ** DRT PLTHT.V13 Construct 135.01 136.86 −1.850.01485240 ** DRT LFN.V13 DTP4-L13 12.90 12.97 −0.07 0.39420034 DRTLFN.V13 DTP4-L16 12.87 12.80 0.07 0.39420034 DRT LFN.V13 DTP4-L17 12.8012.97 −0.17 0.03413080 ** DRT LFN.V13 Construct 12.86 12.91 −0.060.21929525 DRT PLTHT.V16 DTP4-L13 173.44 174.19 −0.75 0.60570222 DRTPLTHT.V16 DTP4-L16 172.78 172.69 0.07 0.96121412 DRT PLTHT.V16 DTP4-L17172.46 174.43 −1.97 0.16863300 DRT PLTHT.V16 Construct 172.89 173.77−0.88 0.28458043 DRT DUALEX.CHL DTP4-L13 41.39 42.09 −0.70 0.55913823DRT DUALEX.CHL DTP4-L16 41.68 41.04 0.65 0.58371725 DRT DUALEX.CHLDTP4-L17 41.81 41.80 0.01 0.99403591 DRT DUALEX.CHL Construct 41.6341.64 −0.01 0.98483459 DRT DUALEX.FLV DTP4-L13 0.73 0.81 −0.080.10497460 DRT DUALEX.FLV DTP4-L16 0.72 0.86 −0.14 0.00384718 ** DRTDUALEX.FLV DTP4-L17 0.78 0.72 0.06 0.21800652 DRT DUALEX.FLV Construct0.74 0.80 −0.05 0.05661318 * DRT DUALEX.NBI DTP4-L13 59.84 54.78 5.070.15566030 DRT DUALEX.NBI DTP4-L16 62.23 53.77 8.47 0.01832608 ** DRTDUALEX.NBI DTP4-L17 57.05 63.58 −6.54 0.07037471 * DRT DUALEX.NBIConstruct 59.71 57.38 2.33 0.25803990 DRT RWC DTP4-L13 63.07 60.05 3.020.00001012 ** DRT RWC DTP4-L16 63.26 60.92 2.34 0.00167083 ** DRT RWCDTP4-L17 60.35 63.71 −3.37 0.00005237 ** DRT RWC Construct 62.22 61.560.66 0.11241576 DRT PLTHT.R1 DTP4-L13 240.17 238.29 1.88 0.26645568 DRTPLTHT.R1 DTP4-L16 238.63 237.80 −1.18 0.46424610 DRT PLTHT.R1 DTP4-L17236.32 241.45 −5.13 0.00738054 ** DRT PLTHT.R1 Construct 237.70 239.18−1.48 0.24612187 DRT EARHT DTP4-L13 108.91 108.11 0.79 0.82357635 DRTEARHT DTP4-L16 112.05 115.87 −3.82 0.25470767 DRT EARHT DTP4-L17 110.96111.45 −0.49 0.89285465 DRT EARHT Construct 110.64 111.81 −1.170.56248804 DRT LFN DTP4-L13 18.77 18.90 −0.13 0.36030764 DRT LFNDTP4-L16 18.77 18.97 −0.20 0.15503518 DRT LFN DTP4-L17 18.77 18.87 −0.100.47612562 DRT LFN Construct 18.77 18.91 −0.14 0.07918268 * DRT EARLPDTP4-L13 11.44 11.66 −0.21 0.25184046 DRT EARLP DTP4-L16 11.30 11.62−0.31 0.08839970 * DRT EARLP DTP4-L17 11.45 11.49 −0.04 0.83236924 DRTEARLP Construct 11.40 11.59 −0.19 0.07795055 * DRT DWMAIN.R1 DTP4-L13128.67 135.77 −7.11 0.00067236 ** DRT DWMAIN.R1 DTP4-L16 127.87 130.60−2.73 0.22100772 DRT DWMAIN.R1 DTP4-L17 124.70 128.09 −3.38 0.17081765DRT DWMAIN.R1 Construct 127.08 131.49 −4.41 0.00111012 ** DRT DWTIL.R1DTP4-L13 6.43 3.90 2.53 0.09173020 ** DRT DWTIL.R1 DTP4-L16 6.31 3.043.27 0.02219856 ** DRT DWTIL.R1 DTP4-L17 9.25 2.41 6.84 0.00001569 **DRT DWTIL.R1 Construct 7.33 3.12 4.21 0.00000292 ** DRT DWVEG.R1DTP4-L13 135.77 138.70 −2.92 0.44895425 DRT DWVEG.R1 DTP4-L16 135.94131.22 4.72 0.22226946 DRT DWVEG.R1 DTP4-L17 133.02 130.33 2.680.50819162 DRT DWVEG.R1 Construct 134.91 133.42 1.49 0.52385836 DRTDWEAR.R1 DTP4-L13 1.28 1.30 −0.02 0.94930818 DRT DWEAR.R1 DTP4-L16 1.441.02 0.42 0.14805885 DRT DWEAR.R1 DTP4-L17 1.70 0.92 0.78 0.01358393 **DRT DWEAR.R1 Construct 1.48 1.08 0.40 0.03074121 ** DRT DWTOT.R1DTP4-L13 136.64 139.84 −3.19 0.41581367 DRT DWTOT.R1 DTP4-L16 137.62132.58 5.04 0.20337115 DRT DWTOT.R1 DTP4-L17 134.83 131.47 3.350.41814064 DRT DWTOT.R1 Construct 136.36 134.63 1.73 0.46821874 DRT SHEDDTP4-L13 61.80 61.97 −0.17 0.57903541 DRT SHED DTP4-L16 61.97 82.43−0.47 0.12144252 DRT SHED DTP4-L17 62.63 61.83 0.80 0.00835084 ** DRTSHED Construct 62.13 62.08 0.06 0.74866113 DRT SILK DTP4-L13 65.11 65.66−0.55 0.23958634 DRT SILK DTP4-L16 65.20 65.29 −0.10 0.83421664 DRT SILKDTP4-L17 64.98 66.26 −1.28 0.01041071 ** DRT SILK Construct 65.09 65.73−0.64 0.01991307 ** DRT ASI DTP4-L13 3.38 3.88 −0.50 0.34118899 DRT ASIDTP4-L16 3.51 2.89 0.62 0.22779519 DRT ASI DTP4-L17 2.48 4.64 −2.170.00014762 ** DRT ASI Construct 3.12 3.80 −0.68 0.02729734 ** DRTGR.V6V10 DTP4-L13 5.39 5.50 −0.11 0.13336177 DRT GR.V6V10 DTP4-L16 5.345.40 −0.06 0.41283559 DRT GR.V6V10 DTP4-L17 5.14 5.42 −0.28 0.00013367** DRT GR.V6V10 Construct 5.29 5.44 −0.15 0.00056568 ** DRT GR.V10V13DTP4-L13 5.35 5.55 −0.19 0.14897867 DRT GR.V10V13 DTP4-L16 5.43 5.270.15 0.26051959 DRT GR.V10V13 DTP4-L17 5.59 5.44 0.15 0.25486733 DRTGR.V10V13 Construct 5.46 5.42 0.04 0.62904773 DRT YL.7.15 DTP4-L13 6.987.11 −0.13 0.19340561 DRT YL.7.15 DTP4-L16 6.74 6.94 −0.20 0.04179433 **DRT YL.7.15 DTP4-L17 6.75 6.98 −0.23 0.01922373 ** DRT YL.7.15 Construct6.82 7.01 −0.19 0.00123685 ** DRT YL.8.1 DTP4-L13 9.88 10.41 −0.530.05275571 * DRT YL.8.1 DTP4-L16 9.96 9.98 −0.02 0.94170108 DRT YL.8.1DTP4-L17 9.70 9.97 −0.27 0.33026794 DRT YL.8.1 Construct 9.85 10.12−0.27 0.09054654 * DRT YL.8.11 DTP4-L13 10.99 11.76 −0.77 0.00606165 **DRT YL.8.11 DTP4-L16 10.84 10.87 −0.04 0.89082227 DRT YL.8.11 DTP4-L1710.44 10.95 −0.51 0.07267146 * DRT YL.8.11 Construct 10.75 11.19 −0.440.00749806 ** DRT DWVEG.R6 DTP4-L13 114.68 118.93 −4.26 0.610440457 DRTDWVEG.R6 DTP4-L16 119.38 120.98 −1.60 0.848086748 DRT DWVEG.R6 DTP4-L17120.64 146.04 −25.40 0.005617906 ** DRT DWVEG.R6 Construct 118.23 128.65−10.42 0.037370754 ** DRT ROW.1 DTP4-L13 15.56 15.07 0.49 0.392964042DRT ROW.1 DTP4-L16 15.33 16.12 −0.78 0.158014994 DRT ROW.1 DTP4-L1715.12 15.00 0.12 0.843163311 DRT ROW.1 Construct 15.34 15.40 −0.060.858801963 DRT DWK DTP4-L13 95.65 79.98 15.67 0.151305864 DRT DWKDTP4-L16 93.74 84.95 8.78 0.393267032 DRT DWK DTP4-L17 84.86 62.85 22.000.049180594 ** DRT DWK Construct 91.42 75.93 15.49 0.013697301 ** DRTDWCOB DTP4-L13 15.50 14.52 0.98 0.406520641 DRT DWCOB DTP4-L16 15.8315.23 0.61 0.585311369 DRT DWCOB DTP4-L17 15.67 15.00 0.66 0.580121718DRT DWCOB Construct 15.67 14.92 0.75 0.265159919 DRT DWEAR DTP4-L13110.98 94.32 16.66 0.149473434 DRT DWEAR DTP4-L16 109.53 100.18 9.360.390106055 DRT DWEAR DTP4-L17 100.57 77.88 22.69 0.055285154 DRT DWEARConstruct 107.03 90.79 16.24 0.014608231 ** DRT KN DTP4-L13 373.56282.57 90.99 0.042767336 ** DRT KN DTP4-L16 341.47 324.97 16.500.696092766 DRT KN DTP4-L17 315.12 228.80 86.32 0.058673186 DRT KNConstruct 343.38 278.78 64.60 0.012066189 ** DRT X100KW DTP4-L13 26.0827.58 −1.50 0.156126321 DRT X100KW DTP4-L16 28.07 26.87 1.19 0.228474518DRT X100KW DTP4-L17 27.11 26.80 0.32 0.768899771 DRT X100KW Construct27.09 27.08 0.00 0.994088329 DRT DWTOT DTP4-L13 218.34 204.63 13.700.25823758 DRT DWTOT DTP4-L16 219.85 216.85 3.00 0.803698091 DRT DWTOTDTP4-L17 212.97 209.09 3.88 0.767715805 DRT DWTOT Construct 217.05210.19 6.86 0.339825972 DRT HI DTP4-L13 0.39 0.31 0.08 0.147886079 DRTHI DTP4-L16 0.36 0.37 0.00 0.937885549 DRT HI DTP4-L17 0.34 0.21 0.130.025392684 ** DRT HI Construct 0.36 0.30 0.07 0.033449426 ** (WW = wellwatered; DRT = drought stressed; ** p value < 0.05; * p value < 0.1))

Example 39 Profile HMM Specific to DTP4

Profile HMMs are statistical models of multiple sequence alignments, oreven of single sequences. They capture position-specific informationabout how conserved each column of the alignment is, and which residuesare likely.

Description:

HMMER® (biosequence analysis using profile hidden Markov models) is usedto search sequence databases for homologs of protein sequences, and tomake protein sequence alignments. HMMER® can be used to search sequencedatabases with single query sequences, but it becomes particularlypowerful when the query is a multiple sequence alignment of a sequencefamily. HMMER® makes a profile of the query that assigns aposition-specific scoring system for substitutions, insertions, anddeletions. HMMER® profiles are probabilistic models called “profilehidden Markov models” (profile HMMs) (Krogh et al., 1994, J. Mol. Biol.,235:1501-1531; Eddy, 1998, Curr. Opin. Struct. Biol., 6:361-365.; Durbinet al., Probabilistic Models of Proteins and Nucleic Acids. CambridgeUniversity Press, Cambridge UK. 1998, Eddy, Sean R., March 2010, HMMERUser's Guide Version 3.0, Howard Hughes Medical Institute, Janelia FarmResearch Campus, Ashburn Va., USA; US patent publication No.US20100293118). Compared to BLAST, FASTA, and other sequence alignmentand database search tools based on older scoring methodology, HMMER®aims to be significantly more accurate and more able to detect remotehomologs, because of the strength of its underlying probability models.

Method for Creating Profile HMMs Specific to DTP4 Gene Family Step1:Identification of Homologs of AT-DTP4:

Homologs for AT-CXE20 were identified by querying protein sequence ofAT-DTP4 using BLAST and Jackhammer within an in house database ofprotein sequences generated by compilation of protein sequences fromUniProt and translated ORFs from various plant genomes that wereretrieved from NCBI and internal sequencing cDNA sequencing data.Homologs thus identified were aligned using the software MUSCLE (Edgar,Robert C. (2004), Nucleic Acids Research 19; 32(5):1792-7) using theMEGA6 program (Phylogenetic and molecular evolutionary analyses wereconducted using MEGA version 6 (Tamura K., et al (2013) Mol. Biol. Evol.30 (12): 2725-2729). Phylogenetic analysis was done with the MEGA6program, and the Maximum Likelihood method (Jones D. T., et al (1992).Comp Appl Biosci 8: 275-282; Tamura K., et al (2013) Mol. Biol. Evol. 30(12): 2725-2729).

Branches of the resulting tree were annotated according to Marshall etal J Mol Evol (2003) 57:487-500. Utilizing the Marshall nomenclature, asubset of genes from CXE tree, Type II, Type IV, Type V, and Type VIwere isolated and realigned. A new Maximum Likelihood tree was builtusing just these proteins.

Step 2: Identify and Realign Type II Carboxylesterases

Proteins specific to the Type II lead branch were realigned and a newtree was built with the same process as step 1. Proteins from the newType II specific tree were then picked based on the branching pattern inorder to get one protein per sub branch. These proteins, SEQ ID NOS:18,29, 33, 45, 47, 53, 55, 61,64, 65, 77, 78, 101, 103, 105, 107, 111, 115,131, 132, 135, 137, 139, 141, 144, 433, 559 and 604, were realigned andused for the HMM build in step 3.

Step 3: Creating profile HMM for DTP4

HMMbuild module of HMMER® 3.0 was used to create a profile HMM for DTP4based on Multiple Sequence Alignment (MSA) of homologs of AT-CXE20.

Step 4: Using Profile to Search Protein Database

Profile HMM created was queried in a database of protein sequencesdescribed in Step 1. Hits retrieved were further examined as describedin Step 5.

Step5: Determining Specificity of Profile to Identify DTP4 RelatedProtein Sequences

All protein sequences that matched the profile HMM of CXE20 with anE-value of less than 0.001 over at least 80% length of the HMM profilewere regarded as statistically significant and corresponding to genefamily. Since all statistically significant protein hits obtained aremembers of CXE20 gene family, it is suggested that profile HMM for CXE20described here is specific to prioritize ranking of the Type IIcarboxylesterases, and identify other members of the carboxylesterasefamily. The HMM profile for CXE20 family is shown in the appended Table18.

Example 40 Targeted Regulation or Mutagenesis of an Endogenous DTP4 Gene

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences, thereby leading tochanges in either the expression of encoded mRNAs or the amino acidsequence of the encoded polypeptide e.g., DTP4, resulting in alterationof the biological activity of the mRNA or protein, respectively, orboth. See for example methods described in U.S. patent application Ser.No. 14/463,687 filed on Aug. 20, 2014, incorporated by reference in itsentirety herein. Thus, variant nucleic acid molecules can be created byintroducing one or more nucleotide substitutions, additions and/ordeletions into the corresponding nucleic acid sequence or surroundingsequences disclosed herein. Such variant nucleic acid sequences are alsoencompassed by the present disclosure.

Variant nucleic acid sequences can be made by introducing sequencechanges randomly along all or part of the genic region, including, butnot limited to, chemical or irradiation mutagenesis andoligonucleotide-mediated mutagenesis (OMM) (Beetham et al. 1999; Okuzakiand Toriyama 2004). Alternatively or additionally, sequence changes canbe introduced at specific selected sites using double-strand-breaktechnologies such as ZNFs, custom designed homing endonucleases, TALENs,CRISPR/CAS (also referred to as guide RNA/Cas endonuclease systems (U.S.patent application Ser. No. 14/463,687 filed on Aug. 20, 2014), or otherprotein and/or nucleic acid based mutagenesis technologies. Theresultant variants can be screened for altered activity. It will beappreciated that the techniques are often not mutually exclusive.Indeed, the various methods can be used singly or in combination, inparallel or in series, to create or access diverse sequence variants.

TABLE 18 HMM profile HMMER3/b [3.0 | March 2010] NAMECXE20_TypeIIBranch_limit_one_perSubBranch LENG 326 ALPH amino RF no CSno MAP yes DATE Thu Nov 13 16:23:57 2014 NSEQ 28 EFFN 1.124512 CKSUM701189305 STATS LOCAL MSV −11.1717 0.70062 STATS LOCAL VITERBI −12.01970.70062 STATS LOCAL FORWARD −5.8925 0.70062 HMM A C D E F G H I K L Mm−>m m−>i m−>d i−>m i−>i d−>m d−>d COMPO 2.50926 4.25552 2.8369 2.711393.33164 2.91186 3.58549 2.84504 2.73821 2.42491 3.61156 2.68593 4.422742.77541 2.73137 3.46258 2.40556 3.72518 3.29342 2.67758 2.69305 4.243630.84551 1.24233 1.26602 2.01075 0.14374 0 * 1 2.51668 4.86384 2.873542.09458 4.13606 2.98579 3.57833 3.56272 2.2325 3.13434 3.60294 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0295 3.93385 4.65619 0.61958 0.77255 0.70739 0.67911 2 2.399924.84776 2.4917 2.27086 4.10996 3.22751 3.58926 3.53813 2.37511 3.002623.41645 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.02778 3.99313 4.71547 0.61958 0.77255 0.675470.71114 3 2.49757 4.94613 2.67419 2.21463 4.23287 3.19058 3.578863.67816 2.16759 3.1002 3.53885 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02645 4.04144 4.763790.61958 0.77255 0.52031 0.90223 4 2.51011 4.75462 2.90058 2.394193.98528 3.15565 3.65758 3.39418 2.47537 3.01332 3.00755 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.02353 4.15705 4.8794 0.61958 0.77255 0.60407 0.79094 5 2.49208 4.735143.04507 2.37102 3.95612 3.44744 3.50593 3.18094 2.41281 2.85253 3.811562.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.02353 4.15705 4.8794 0.61958 0.77255 0.56577 0.83915 62.54242 4.91918 2.53966 2.21759 4.19078 3.41485 3.61804 3.62671 2.3943.04389 3.75267 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.02307 4.17665 4.899 0.61958 0.772550.5825 0.81758 7 2.54402 4.87337 2.78891 2.41637 4.13149 3.19872 3.452353.55859 2.26532 2.7798 3.71016 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02307 4.17665 4.8990.61958 0.77255 0.53702 0.87824 8 2.37704 4.51025 3.25209 2.703893.76346 3.45872 3.78012 2.80156 2.67734 2.81709 3.43996 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.03938 4.19951 3.74604 0.61958 0.77255 0.55617 0.85191 9 2.51441 3.93573.19229 2.63786 3.54278 3.46497 3.73718 3.17037 2.51696 2.74557 3.690242.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.02292 4.18305 4.9054 0.61958 0.77255 0.57526 0.82681 102.45991 4.53639 3.21275 2.65303 3.71899 3.5 3.73377 2.55299 2.438962.77209 3.38373 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.02292 4.18305 4.9054 0.61958 0.772550.46321 0.99226 11 2.36766 4.34189 3.22428 2.8129 3.14532 3.586983.84004 2.60558 2.86376 2.50783 2.86048 2.6862 4.42232 2.77526 2.731073.4636 2.40519 3.72501 3.29341 2.6774 2.69361 4.24673 0.16912 1.906514.96231 0.84355 0.56244 0.47318 0.97557 12 2.74238 4.13315 1.607172.15785 4.48655 3.39894 3.68695 3.9551 2.42318 3.47378 4.25285 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 13 2.472984.43247 3.40454 2.87901 3.47482 3.46218 3.89595 3.06143 2.84119 2.410193.66713 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 14 2.73996 4.49232 3.23879 2.94928 2.69271 3.65463 3.791893.01588 2.93588 2.69147 3.60827 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 15 2.48769 5.06628 2.55476 2.178193.95365 3.41774 3.61498 3.83898 1.85574 3.17541 4.11754 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.04727 4.25552 3.44244 0.61958 0.77255 0.48576 0.9551 16 2.484054.22073 3.7005 3.12776 2.55502 3.64194 3.27538 2.48512 3.05421 2.24443.32947 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.02185 4.2301 4.95245 0.61958 0.77255 0.518830.90441 17 3.13366 3.92313 4.83811 4.25221 2.78383 4.35472 4.663442.16434 4.11395 0.96672 2.5582 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02185 4.2301 4.952450.61958 0.77255 0.51883 0.90441 18 2.71352 5.1651 2.71849 2.3519 4.497242.7822 3.63847 3.96317 1.91694 3.45632 4.22248 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.021854.2301 4.95245 0.61958 0.77255 0.46563 0.98816 19 3.29145 4.5849 5.091444.5213 2.86124 4.60569 4.95187 1.22139 4.39349 1.40019 2.67523 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 20 2.50714.38003 3.4532 2.88775 3.52986 3.58488 3.61016 2.669 2.73495 2.59823.48719 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 21 2.62803 4.75161 3.07898 2.46141 3.97002 3.47631 3.301313.08914 2.40303 2.40822 3.82851 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 22 2.90861 5.22831 2.27588 2.372034.69297 3.33461 3.92914 4.19276 2.88341 3.76602 4.61584 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 23 2.20575 4.381273.4243 3.21249 4.49761 3.11767 4.35898 3.90077 3.31371 3.60277 4.450762.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 243.09358 5.68885 1.00725 2.21734 4.97028 3.34539 3.9055 4.52687 2.937394.0077 4.8622 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 25 2.33146 4.60859 2.75755 2.86722 4.63275 0.942014.25777 4.06508 3.27582 3.7369 4.58297 2.68619 4.42226 2.7751 2.731243.46355 2.40514 3.72495 3.29355 2.67742 2.69356 4.24691 0.07416 2.739694.97786 0.42151 1.06727 0.48576 0.9551 26 2.38454 4.33004 3.595653.28357 4.42041 3.11148 4.34501 3.7987 3.29928 3.51775 4.35848 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 27 3.031473.82107 4.83556 4.26047 2.92881 4.29441 4.66038 1.84664 4.11892 1.295193.17251 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 28 2.55623 3.34433 4.00897 3.51787 3.75796 3.44628 4.318122.50251 3.39914 2.70649 3.70826 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 29 2.93208 3.75576 3.62881 2.97162 4.32043.65869 3.81008 3.6711 2.13704 3.26094 4.14624 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.048394.25552 3.40981 0.61958 0.77255 0.48576 0.9551 30 2.54708 4.909622.76214 2.41345 3.89732 3.43334 3.29521 3.6061 2.40981 2.90911 3.972812.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.02188 4.22901 4.95136 0.61958 0.77255 0.5202 0.90239 312.60652 4.28625 3.55578 2.98681 3.1365 3.22339 3.86541 2.63665 2.933032.10958 3.39293 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.02188 4.22901 4.95136 0.61958 0.772550.5202 0.90239 32 2.57655 4.18133 2.6128 2.24332 4.194 3.27508 3.628243.62945 2.21816 3.07846 3.68912 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.05407 4.22901 3.268330.61958 0.77255 0.5202 0.90239 33 2.43549 4.1946 3.49837 3.13013 2.62663.62993 3.34687 2.40398 2.96131 2.1902 3.30392 2.6862 4.42227 2.775212.73125 3.46318 2.40515 3.72496 3.29356 2.67743 2.69357 4.24692 0.142732.07379 4.91987 0.31478 1.30915 0.44282 1.02785 34 2.57139 4.504563.38791 3.15943 4.24021 3.24832 4.26813 3.7144 3.16197 3.19789 4.346982.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 352.62406 4.0722 3.00641 2.46344 3.90714 3.48517 3.51516 3.1024 2.42372.603 3.78153 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 36 2.63863 4.21241 4.10815 3.53968 3.37574 3.828524.18783 2.15385 3.43691 1.94731 3.33898 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 37 2.44402 4.92605 2.829992.26477 4.19092 3.43704 3.65541 3.62256 2.44347 3.19924 3.99706 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 38 2.061424.68315 2.99137 2.57347 3.92558 3.46724 3.72229 3.17286 2.49658 2.754943.80199 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 39 2.43112 4.94294 2.73453 2.33796 4.24985 3.41808 3.658863.68913 2.44198 3.25309 4.04181 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 40 2.61022 5.0076 2.54438 2.20551 4.314132.92472 3.69421 3.45771 2.50376 3.31933 4.11535 2.68613 4.42227 2.775122.73118 3.46356 2.40515 3.72497 3.29356 2.67743 2.69357 4.24692 0.18591.98299 3.44244 0.28909 1.38209 0.48576 0.9551 41 2.50594 5.129072.16441 2.17619 4.44596 3.24587 3.63132 3.91774 2.40334 3.26819 4.185182.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.04271 4.2301 3.6025 0.61958 0.77255 0.46563 0.98816 422.43273 4.45224 3.32672 2.67828 3.46154 3.19626 3.57269 2.88541 2.731362.37107 3.55001 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.02173 4.23564 4.95798 0.61958 0.772550.51178 0.91485 43 2.53076 4.2533 2.78041 2.303 3.98666 3.46117 3.6782.95489 2.49702 3.01573 3.84078 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.14374 4.23564 2.125110.61958 0.77255 0.51178 0.91485 44 2.41809 4.60677 3.10443 2.576793.89136 3.18884 3.2356 3.2878 2.56095 2.69666 3.77607 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.02452 4.11641 4.83876 0.61958 0.77255 0.64612 0.74249 45 2.468124.8969 2.63698 2.27415 4.17091 2.73607 3.61436 3.60494 2.39812 3.086923.97031 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.04517 4.11641 3.58037 0.61958 0.77255 0.646120.74249 46 2.3075 4.66654 3.05527 2.40601 3.89215 3.21979 3.6661 3.032092.42765 2.7747 3.76439 2.68602 4.42237 2.77515 2.73105 3.46366 2.405163.72507 3.29344 2.67748 2.69367 4.24702 0.22448 1.64512 4.81861 1.221980.34906 0.51688 0.90728 47 2.49806 4.46973 3.28676 2.73345 3.687823.24592 3.7795 3.05723 2.69223 2.37261 3.61507 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.023014.17918 4.90153 0.61958 0.77255 0.57965 0.8212 48 1.98571 4.237933.80646 3.27496 3.55955 3.53029 4.10193 2.42909 3.20483 2.56808 3.513922.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.02301 4.17918 4.90153 0.61958 0.77255 0.53544 0.8804749 2.52321 4.15057 3.86583 3.29091 2.86312 3.67168 3.42234 2.588523.19417 1.74174 3.2549 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0225 4.20135 4.92369 0.619580.77255 0.44534 1.02334 50 2.57415 3.70327 3.54281 2.99502 2.885573.50393 3.67525 2.93372 2.94353 2.64768 3.53896 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 51 3.03784 5.201923.34441 2.79178 4.67364 3.6687 3.73953 4.05042 1.01628 3.51995 4.370092.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 523.45489 5.58801 0.49459 2.66181 5.02904 3.55014 4.33669 4.71613 3.502914.26044 5.2638 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 53 3.2969 4.54806 5.17406 4.66171 3.64699 4.76175 5.303351.05901 4.56121 1.86412 3.42387 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 54 2.51523 4.42651 3.44965 2.991163.99447 3.31959 4.03957 2.9669 2.94654 3.03715 3.92065 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 55 3.09752 4.457384.74599 4.17112 3.20323 4.31658 4.61812 1.7523 4.02848 1.12071 3.090442.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 562.95093 5.36942 2.06163 2.30169 4.78524 3.33332 3.90261 4.31652 2.882643.84293 4.68099 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 57 2.49505 5.01318 2.73197 2.37818 4.30747 3.424833.31799 3.4409 2.18505 3.29665 4.07007 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 58 2.32785 5.06295 2.5553 2.145214.36867 3.41263 3.63946 3.82674 2.41158 3.24953 4.12734 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 59 2.58408 5.047572.91658 2.41742 4.36735 3.44485 3.28417 3.81115 1.88888 3.34431 4.12892.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 602.67551 5.11786 2.86099 2.2947 4.44275 2.77213 3.62687 3.90806 2.050613.41107 4.17233 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 61 2.6752 4.38485 3.94962 3.47431 3.78519 3.58218 4.360242.42683 3.36171 2.60602 3.69799 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 62 2.74023 4.27007 3.95316 3.395082.21757 3.24876 3.88109 2.74526 3.28785 2.45056 3.38906 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 63 2.47751 4.323354.57047 3.99043 2.93453 4.13362 4.46921 2.15166 3.86231 1.36854 2.882492.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 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2.926662.38657 4.20323 2.98808 3.42033 3.6382 2.19866 3.04102 3.99042 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.02431 4.12486 4.84721 0.61958 0.77255 0.4013 1.107 78 3.194125.32193 3.68555 2.93841 4.78873 3.82072 3.70741 4.11892 1.10927 3.267754.40561 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 79 3.53779 4.88359 4.9722 4.50399 3.18088 4.67427 5.01813 2.254764.25136 0.62548 3.04227 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.619580.77255 0.48576 0.9551 80 3.34403 5.01806 3.99464 3.90067 4.835313.68485 4.93048 4.50058 3.99508 4.07601 5.15317 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 81 3.33414 4.600315.16347 4.62514 3.43761 4.73363 5.17762 1.37077 4.50893 1.24191 3.217412.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 823.292 4.54513 5.17125 4.65208 3.60993 4.74672 5.26682 1.17401 4.550491.71895 3.38924 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 83 3.28325 4.53586 5.16709 4.64982 3.63354 4.742725.27171 1.25881 4.55018 1.76836 3.41367 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 84 3.91195 5.12186 4.762064.46281 1.66798 4.58974 2.86357 3.73412 4.30503 3.04323 4.28664 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 85 3.245844.59746 4.64092 4.15596 1.14932 4.26725 3.8925 2.65965 4.00611 2.166453.48035 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 86 3.5141 5.23055 3.59545 3.41016 3.63357 3.83874 0.54184 4.279583.24679 3.71577 4.8026 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.619580.77255 0.48576 0.9551 87 2.48654 4.47887 3.1466 3.03623 4.63948 0.916694.34061 4.15339 3.33413 3.79889 4.62401 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 88 2.43732 4.38898 3.43238 3.31424.67367 0.85033 4.50634 4.18462 3.54341 3.85592 4.68173 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 89 2.48302 4.425723.42777 3.33446 4.68012 0.75761 4.53494 4.21053 3.57682 3.8896 4.731392.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 903.45123 4.74457 4.76377 4.34485 0.9128 4.38 3.72076 3.13876 4.175262.53937 3.15414 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 91 3.27809 4.52947 5.16227 4.66292 3.73986 4.754695.34698 1.28533 4.56683 2.11511 3.51654 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 92 3.31181 4.61513 5.096934.51309 2.42796 4.58582 4.89103 1.93601 4.37544 0.99721 2.6995 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 93 2.424892.89568 4.26787 3.67504 2.35307 3.75528 4.05709 2.42 3.52393 1.918893.16797 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 94 2.6689 4.76953 3.08531 2.65838 4.22485 3.39832 3.53907 3.679222.56446 3.27754 4.10936 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.619580.77255 0.48576 0.9551 95 1.24467 4.2906 3.79855 3.36088 3.92698 3.313434.27409 2.99526 3.31074 2.92265 3.86557 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 96 1.81174 4.97888 2.279922.35233 4.35005 3.38339 3.72643 3.79388 2.54816 3.35863 4.1546 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 97 2.501494.6684 3.00833 2.57649 3.68742 3.48823 3.33848 3.26916 2.56256 2.839783.38488 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.04041 4.25552 3.67217 0.61958 0.77255 0.485760.9551 98 2.28347 4.59251 3.18671 2.62694 3.57625 3.07578 3.730093.16659 2.54968 2.58303 3.68484 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02171 4.23681 4.959160.61958 0.77255 0.51028 0.91711 99 2.46026 4.19767 3.73923 3.164793.01682 3.42545 3.92465 2.28309 3.09077 2.25911 2.84623 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.02171 4.23681 4.95916 0.61958 0.77255 0.47073 0.97962 100 2.645393.49328 3.50742 3.34387 1.72824 3.7061 3.95929 2.55477 3.25032 2.364673.29097 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 101 2.92034 5.26854 2.37287 2.36847 4.40694 3.42588 1.409364.02235 2.63506 3.55809 4.39678 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 102 2.66189 5.14316 2.13318 2.13532 4.4723.10674 3.61773 3.94939 2.17841 3.31547 4.19091 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 103 2.33684 3.918953.56873 3.00135 2.29386 3.60441 3.55768 2.7882 2.83109 2.44158 3.405522.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 1043.10601 0.41162 4.68435 4.52332 4.45138 3.63217 5.08906 3.68031 4.361553.56769 4.70691 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 105 2.19908 4.81464 3.03062 2.39845 3.6726 3.308573.67017 3.47442 2.37197 3.0811 3.89602 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 106 2.31515 5.06582 2.548612.23118 4.37887 3.41569 3.60955 3.84293 2.30627 3.22652 4.11559 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 107 2.544524.1425 4.05777 3.47337 2.99581 3.75268 4.05102 2.30514 3.18757 2.026392.03451 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 108 0.94419 4.26429 3.70371 3.42811 4.51236 2.74569 4.461113.92542 3.4878 3.62397 4.43848 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 109 2.13381 4.996 2.60313 2.33449 4.291093.4252 3.63143 3.73807 2.24504 3.28346 4.06117 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 110 2.34793 5.137792.57319 1.98404 4.46817 3.40727 3.06308 3.94571 2.21015 3.43577 4.184452.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 1112.4266 4.14415 4.24471 3.66152 2.94245 3.83706 4.16521 2.04202 3.535121.63873 3.22367 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 112 2.6543 4.86037 2.99217 2.41084 4.11197 3.3 3.517983.52965 2.46142 2.9452 3.94192 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 113 1.16524 4.29772 3.74505 3.277023.94663 3.29199 4.21077 3.09091 3.21612 2.83092 3.88199 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 114 3.291664.54773 5.16554 4.6417 3.57716 4.73506 5.2389 1.24662 4.53759 1.611053.35831 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 115 3.22756 4.52084 5.00047 4.48885 3.67836 4.61741 5.173021.67203 4.37376 2.17264 3.20592 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 116 2.14283 4.32426 4.43999 3.879263.22525 4.1091 4.49504 2.0212 3.77574 1.90406 3.34717 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 117 2.550964.46721 3.59442 3.44696 4.49053 3.19268 4.53558 4.07531 3.54843 3.770964.67466 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 118 2.66485 4.31652 4.17925 3.64964 3.49325 3.8828 4.358772.25778 3.53449 1.91036 3.41621 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 119 2.92591 5.47948 1.92947 1.302034.7768 3.36561 3.44454 4.2859 2.70549 3.77178 4.5739 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 120 3.847855.17916 4.42837 4.24429 2.41835 4.29566 3.86875 3.81079 4.093 3.16124.4472 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 121 3.49583 5.27252 4.0307 3.48176 4.72839 3.88626 4.181 4.313062.49598 3.75907 4.76361 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.619580.77255 0.48576 0.9551 122 3.05693 4.63825 4.01003 3.65419 3.348383.91996 4.36755 2.68232 3.43279 0.82632 3.47935 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 123 0.78484 3.635034.02275 3.66567 4.30301 3.09883 4.51946 3.5561 3.5848 3.3719 4.253412.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 1243.34403 5.01806 3.99464 3.90067 4.83531 3.68485 4.93048 4.50058 3.995084.07601 5.15317 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 125 3.01119 5.30786 2.5308 0.89517 4.7118 3.40936 3.954194.09498 2.83177 3.71332 4.62081 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 126 2.87112 5.22503 2.55133 2.387024.46183 3.44072 1.60056 3.97854 2.36584 3.50913 4.32976 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 127 3.3081 5.331523.78728 3.07699 4.83307 3.86274 3.77499 4.17432 1.79447 3.59147 4.491112.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 1283.6167 4.975 4.80486 4.46322 3.33052 4.46513 5.00174 2.57039 4.228880.51488 3.26288 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 129 3.34403 5.01806 3.99464 3.90067 4.83531 3.684854.93048 4.50058 3.99508 4.07601 5.15317 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 130 0.57778 4.43246 3.900783.73115 4.4826 3.24313 4.70044 3.66968 3.75883 3.5317 4.534 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 131 1.096644.46203 3.48594 3.0602 3.91557 3.39018 4.07905 3.17677 2.95982 2.896083.4621 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 132 3.94642 5.14309 4.81231 4.55796 1.84154 4.60234 3.609233.63287 4.39292 2.92259 4.22715 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 133 3.00215 5.60916 1.37851 1.565344.89385 3.35789 3.49917 4.41969 2.78481 3.8899 4.70611 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 134 3.454895.58801 0.49459 2.66181 5.02904 3.55014 4.33669 4.71613 3.50291 4.260445.2638 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 135 1.15958 3.63692 3.8944 3.46324 4.20666 1.97236 4.366143.56413 3.41022 3.29846 4.14315 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 136 2.28315 4.21242 3.75361 2.97643.23681 3.66541 3.94331 2.56676 3.10337 2.10265 2.95782 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 137 2.721635.47373 1.79923 1.35158 4.7792 3.36708 3.78704 4.28314 2.68891 3.767114.56605 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 138 0.79209 4.25149 3.78593 3.53079 4.46614 3.06674 4.515553.75432 3.54784 3.55222 4.40622 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 139 3.33894 4.60488 5.16628 4.628013.43382 4.73808 5.18085 1.34876 4.51121 1.24049 3.21303 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 140 2.539824.86299 2.93834 2.35311 4.11108 3.45389 2.91627 3.53257 2.23139 2.518323.36704 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 141 3.14158 3.9883 4.43671 3.94113 2.43765 4.10081 3.888012.89559 3.74377 2.29885 3.57298 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 142 2.6268 4.42605 4.89134 4.361113.66987 4.48221 4.98904 1.32971 4.25959 2.08651 3.49478 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 143 3.252415.39101 3.71565 2.95953 4.91102 3.84901 3.70354 4.22048 1.39736 3.601244.46615 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 144 2.609 5.15173 2.2255 2.1938 4.4729 3.40238 3.63574 3.948452.31029 3.44779 4.2053 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.619580.77255 0.48576 0.9551 145 2.65663 5.02967 2.89993 2.27601 4.327653.06402 3.26627 3.60889 2.38934 3.31486 4.09139 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.207494.25552 1.75337 0.61958 0.77255 0.48576 0.9551 146 1.81814 4.27163.53266 2.9675 3.38925 3.56426 3.84427 2.73772 2.88407 2.44525 3.183212.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.06346 4.07363 3.11296 0.61958 0.77255 0.68685 0.69948147 2.25203 4.55727 2.93689 2.5659 3.74719 3.45084 3.67538 3.132322.54399 2.49491 3.18094 2.68601 4.4222 2.7748 2.73117 3.46379 2.405013.72519 3.2933 2.6776 2.6938 4.24686 0.53954 0.89548 4.7591 1.28960.32211 0.36328 1.18872 148 2.65415 3.18473 2.60629 2.32833 4.192072.21188 3.68897 3.62047 2.40697 3.20657 4.01202 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 149 2.95227 5.51431.51524 1.48816 4.81612 3.37033 3.80443 4.32435 2.7102 3.80498 4.612012.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 1502.64631 5.20955 2.41236 1.91823 4.52272 3.39858 3.67522 3.99815 2.374963.50144 4.2719 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 151 3.2118 4.72114 4.38042 3.9584 2.73383 4.02996 4.034273.0267 3.65069 2.50896 3.74225 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.06553 4.25552 3.011020.61958 0.77255 0.48576 0.9551 152 3.35631 4.65203 5.06637 4.504523.00735 4.66836 4.99974 1.87686 4.38618 0.91426 3.01592 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.02225 4.21224 4.93459 0.61958 0.77255 0.54094 0.87276 153 2.572135.02638 2.98342 2.34543 4.3417 3.45414 3.62375 3.77932 1.801 3.308314.09133 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.02225 4.21224 4.93459 0.61958 0.77255 0.540940.87276 154 2.62432 5.28573 2.00127 1.76343 4.60388 3.37014 3.68234.09412 2.37067 3.57989 4.34911 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02225 4.21224 4.934590.61958 0.77255 0.54094 0.87276 155 2.81824 4.46049 3.7236 3.179062.60538 3.46919 2.83604 2.9695 3.09007 2.34936 3.57361 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.02225 4.21224 4.93459 0.61958 0.77255 0.54094 0.87276 156 1.476094.28772 3.67705 3.2009 3.82281 2.50801 4.13442 2.97689 3.15373 2.710173.76755 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.02225 4.21224 4.93459 0.61958 0.77255 0.540940.87276 157 3.39607 5.53382 0.53169 2.61585 4.96615 3.50591 4.283544.64066 3.43962 4.19216 5.19108 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02225 4.21224 4.934590.61958 0.77255 0.54094 0.87276 158 2.59342 4.18619 4.09052 3.518581.90243 3.78729 4.00775 2.55852 3.40225 1.86364 3.22649 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.02225 4.21224 4.93459 0.61958 0.77255 0.54094 0.87276 159 2.496754.49787 3.13901 2.95607 4.43779 3.14537 4.20364 3.98887 3.13126 3.638564.47861 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.02225 4.21224 4.93459 0.61958 0.77255 0.540940.87276 160 2.8841 5.20295 3.14784 2.51492 4.59649 3.57921 3.649564.00008 1.65277 3.46379 4.2695 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02225 4.21224 4.934590.61958 0.77255 0.54094 0.87276 161 2.6219 1.93898 4.27871 3.760343.51947 3.62944 4.35392 2.36237 3.62209 2.39602 3.48141 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.02225 4.21224 4.93459 0.61958 0.77255 0.45274 1.01028 162 3.629284.89339 4.80118 4.44183 0.97228 4.47872 3.68242 3.26598 4.27962 2.650173.8916 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 163 3.38998 4.66903 5.16045 4.59769 3.27725 4.73483 5.098181.68059 4.47497 0.96503 2.80972 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 164 2.87451 3.82956 4.3442 3.769543.20791 3.99597 4.30617 2.22984 3.62126 1.98337 1.40906 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 165 3.213254.93276 3.96373 3.91957 5.02096 0.32494 4.99846 4.73153 4.15183 4.330015.32112 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 166 2.599 3.61046 3.07338 2.69714 3.77099 3.18004 3.77854 3.050562.67549 2.82596 3.3173 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.619580.77255 0.48576 0.9551 167 2.73773 4.90439 2.63189 2.52675 4.272733.36026 3.2046 3.83506 2.74112 3.42567 4.26347 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 168 1.1864 4.423743.36272 3.00088 4.34072 3.17173 4.14428 3.74875 3.06264 3.41264 4.234862.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 1693.21325 4.93276 3.96373 3.91957 5.02096 0.32494 4.99846 4.73153 4.151834.33001 5.32112 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 170 1.42474 4.31808 3.64137 3.4459 4.60884 1.14769 4.53074.00013 3.57302 3.7257 4.55933 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 171 2.67644 4.75042 2.92595 2.727354.45964 3.28645 4.05372 3.89194 2.9131 3.56299 4.42685 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 172 3.367064.64594 5.13029 4.57809 3.33733 4.72482 5.10688 1.07176 4.44057 1.466442.60671 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 173 1.21161 4.2581 3.83859 3.39579 3.97834 3.25475 4.292463.10257 3.34173 3.01087 3.91839 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 174 3.7362 4.96075 4.86903 4.530571.47683 4.55341 3.65638 3.34755 4.35966 2.21297 3.94457 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 175 2.6582 4.883763.02439 2.47396 3.34774 3.47113 2.5466 3.55648 2.43588 3.14092 3.953522.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 1761.68939 4.30246 3.97245 3.4616 3.68491 3.6115 4.28948 2.46435 3.382142.48409 3.61002 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 177 1.47593 4.35612 3.42796 3.17287 4.52111 1.385864.32882 3.95721 3.31242 3.62019 4.43073 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 178 3.51212 4.80626 5.155444.58484 3.17208 4.80452 5.10508 2.16226 4.44142 0.73151 2.49265 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 179 2.531364.85113 3.15726 2.57206 4.09761 3.5279 3.47496 3.49952 2.26118 2.982813.59713 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 180 1.55978 4.30143 3.68081 3.15647 3.69725 3.43196 4.064032.89411 3.09857 2.73744 3.38369 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 181 1.92279 3.87083 3.73426 3.173743.41056 3.31117 3.96755 2.72845 3.09618 1.82663 3.39405 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 182 2.600495.08818 2.06666 2.0011 4.39685 3.21005 3.63204 3.85978 2.39913 3.259053.71619 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 183 2.22137 4.44866 3.19998 2.7811 3.605 3.55543 3.24533 2.783852.74656 2.14455 3.54717 2.68614 4.42231 2.77504 2.73122 3.4636 2.405123.72481 3.2936 2.67747 2.69361 4.24696 0.2552 1.7373 3.01102 0.669580.71729 0.48576 0.9551 184 3.07385 5.68653 1.03444 1.94309 4.96563.32234 3.87645 4.49781 2.91082 3.98007 4.83283 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.022254.21224 4.93459 0.61958 0.77255 0.54094 0.87276 185 3.04101 4.434944.58213 4.00585 2.96286 4.23225 4.55171 2.17464 3.88822 1.07497 2.894732.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.02225 4.21224 4.93459 0.61958 0.77255 0.54094 0.87276186 2.69084 5.16486 2.48287 1.97237 4.48364 2.92995 3.43767 3.96012.21919 3.45956 4.22139 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.02225 4.21224 4.93459 0.619580.77255 0.45274 1.01028 187 2.37009 4.63835 3.06685 2.7739 4.371552.81692 4.01156 3.78019 2.60484 3.41563 4.2528 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 188 2.8598 4.424644.84922 4.27933 3.33456 4.37237 4.74656 1.70815 4.14979 1.40021 2.518792.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.05262 4.25552 3.29484 0.61958 0.77255 0.48576 0.9551189 2.679 4.4764 3.02533 2.26362 4.27448 3.47031 3.63103 3.44239 1.704683.10517 4.04335 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.02197 4.22487 4.94721 0.61958 0.772550.46175 0.99473 190 3.3305 4.59281 5.1498 4.63453 3.55994 4.758785.26513 0.946 4.51485 1.73726 3.34238 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 191 2.50701 4.89444 2.897532.44443 4.15319 3.45239 3.64741 3.05723 2.2654 3.05665 3.96092 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 192 3.213254.93276 3.96373 3.91957 5.02096 0.32494 4.99846 4.73153 4.15183 4.330015.32112 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 193 3.12638 3.86135 4.81627 4.2433 3.27324 4.37152 4.72982 2.08954.11109 1.0071 2.82613 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.619580.77255 0.48576 0.9551 194 3.0038 4.36972 4.66061 4.12512 3.581443.67296 4.73573 1.13957 4.00155 2.18188 3.45746 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 195 3.52178 4.804965.21863 4.63827 2.89178 4.81139 5.07413 2.12954 4.51017 0.79194 2.149292.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 1962.65124 4.97391 2.76308 2.40569 4.23984 3.43825 2.85334 3.21521 2.406053.24722 4.03573 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 197 2.63916 4.89772 3.08076 2.56258 4.09886 3.510873.07763 3.37711 2.40665 3.14796 3.9932 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 198 2.42207 4.46056 3.526063.21767 4.11878 3.29998 4.27039 3.06106 3.17028 3.10701 4.11363 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 199 3.027954.47618 4.25522 3.73944 1.39929 4.01864 3.12192 2.96004 3.61816 2.4573.26936 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 200 3.58253 4.88367 4.91148 4.50196 0.97239 4.57554 4.180772.69128 4.33385 1.60163 3.3383 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 201 2.36072 4.3236 3.51036 3.287234.60346 1.11963 4.42077 4.04762 3.41991 3.71756 4.52365 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 202 2.392944.48907 3.5425 3.45586 4.72578 0.65686 4.62656 4.18515 3.67885 3.90024.78688 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 203 2.46696 4.3893 3.43591 2.57615 3.54102 3.58601 3.837572.77171 2.72898 2.50837 3.3286 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 204 2.51844 5.16458 2.57081 1.772114.49008 3.40716 3.08068 3.9664 2.14849 3.45936 4.2153 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 205 2.961855.12312 3.2279 2.53337 4.55724 3.61344 3.74682 3.94072 2.1849 3.44874.2943 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 206 2.45604 3.85306 3.70865 3.21226 3.90372 3.28954 4.145522.91497 3.14302 2.96121 3.85001 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 207 2.49946 5.15602 2.56064 1.838764.48321 3.04861 3.62648 3.96107 2.26932 3.45302 4.20576 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 208 1.906024.30156 3.63579 3.40197 4.54129 3.06744 4.46933 3.93437 3.47177 3.6634.49961 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 209 3.14356 5.5952 2.25011 0.81135 4.9211 3.40191 3.97367 4.437122.96883 3.9694 4.85339 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.619580.77255 0.48576 0.9551 210 2.37718 4.16524 3.96337 3.38462 3.284083.7344 4.03432 2.07666 3.00165 1.80389 3.12199 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 211 2.66507 5.211273.24607 2.55853 4.61169 3.63561 3.68164 3.99649 1.81126 3.46672 4.288082.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 2122.63955 4.34791 3.5221 2.96571 3.25704 3.61127 3.85879 2.84249 2.892561.65315 3.4542 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 213 2.22286 4.37073 3.44616 2.54278 3.33023 3.256653.83303 2.568 2.74422 2.58376 3.06763 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 214 2.58034 5.27089 1.929512.25116 4.61496 3.37067 3.75818 4.0935 2.62474 3.60964 4.40214 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 215 3.149395.49467 0.76444 2.4005 5.02233 2.66996 4.09716 4.6021 3.203 4.127925.0223 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 216 2.4882 5.01531 2.94405 2.39158 4.31308 3.05376 3.314243.76108 2.30985 3.29748 4.07233 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 217 2.62023 4.17052 3.85283 3.275543.28797 3.67909 3.56574 2.16353 3.17431 2.36821 3.08873 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 218 3.010143.37642 4.75985 4.17116 2.87029 4.22161 4.54719 2.1197 4.02208 1.083142.73819 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 219 2.56239 4.49247 3.38583 3.07732 4.11907 3.28084 4.161333.54676 3.04325 2.87578 4.17089 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 220 2.6177 4.361 3.51255 3.00735 3.569713.53493 3.93577 2.90704 2.93536 1.51918 3.54787 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 221 2.352 3.4778 3.328742.65448 3.62786 3.54475 3.57969 2.66373 2.73498 2.69126 3.56621 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 222 1.883534.27599 3.5753 3.01584 3.44578 3.58952 3.88616 2.6495 2.95645 2.449863.41792 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 223 2.60199 4.1717 3.2724 2.7169 3.74804 3.20409 3.78685 2.756112.69063 2.70202 3.6666 2.68618 4.42225 2.77519 2.73123 3.46354 2.405133.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.619580.77255 0.48576 0.9551 224 3.13751 5.60334 0.85777 2.30642 4.696863.38916 3.15717 4.47109 3.00456 3.96566 4.85469 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 225 2.57839 4.197523.9564 3.37554 3.10269 3.74858 4.03631 2.54713 3.11039 1.54297 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4.24952 3.65818 3.16064 3.81872 4.12462 2.27674 3.516331.68838 2.09863 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 283 3.49743 4.80833 5.10627 4.53987 3.15607 4.756365.05673 2.29267 4.37793 0.77362 2.16533 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 284 2.67425 5.04246 2.961811.78507 4.34741 3.45204 3.25784 3.46847 2.0028 3.32062 4.09834 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 285 2.184975.09173 2.73433 2.09101 4.40765 2.98645 3.62641 3.87259 2.04636 3.385834.14806 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 286 2.44001 4.00454 3.01117 2.44729 4.26774 3.46287 3.438713.69942 2.00307 3.25166 4.04275 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 287 3.21325 4.93276 3.96373 3.919575.02096 0.32494 4.99846 4.73153 4.15183 4.33001 5.32112 2.68612 4.422262.77521 2.73125 3.46355 2.40508 3.72496 3.29355 2.67735 2.69356 4.246910.04839 3.2103 4.97786 0.76165 0.62904 0.48576 0.9551 288 2.810744.42319 4.66205 4.14475 3.70592 4.29114 4.85958 1.8405 4.02996 2.195643.54246 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 289 2.51151 5.16672 2.39686 2.05227 4.50106 3.40534 2.941253.98238 2.27299 3.46563 4.21348 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 290 2.89766 4.47264 4.68 4.22123 3.736964.21922 4.95695 1.94721 4.07257 2.32057 3.32611 2.68618 4.42225 2.775192.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02134.25552 4.97786 0.61958 0.77255 0.48576 0.9551 291 2.65493 4.71122.97929 2.10081 3.92515 3.49629 3.72875 3.15306 2.41153 2.95305 3.802412.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 2921.93719 4.87093 2.99706 2.17255 4.14496 3.10869 3.668 3.56695 2.389833.15654 3.96244 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 293 2.64823 5.04328 2.8542 2.32909 4.35086 3.43433 2.98113.80385 2.02386 3.32758 3.57284 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 294 2.76882 4.1988 4.32404 3.743721.66172 3.88977 4.1087 2.41086 3.60436 1.91576 2.91596 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 295 2.565645.02622 2.32505 2.23525 4.32416 3.27898 3.619 3.77857 2.31036 3.311144.08034 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 296 2.6898 5.15584 2.16623 1.87611 4.47398 2.99689 3.640793.94745 2.25596 3.44884 4.20944 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 297 2.34498 4.95915 2.53276 2.291174.23019 2.09196 3.67539 3.66339 2.42365 3.2394 4.03998 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 298 2.706364.89473 2.44406 2.49039 4.38194 1.42345 3.60703 3.84057 2.76001 3.434924.25973 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.04054 4.25552 3.6672 0.61958 0.77255 0.485760.9551 299 2.65614 3.18364 4.06911 3.49167 1.97653 3.74134 3.460932.58799 3.14746 2.321 3.25154 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.02171 4.23668 4.959030.61958 0.77255 0.47063 0.97979 300 3.5141 5.23055 3.59545 3.410163.63357 3.83874 0.54184 4.27958 3.24679 3.71577 4.8026 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 301 2.138824.47679 3.34006 2.82079 3.86735 1.78241 3.88707 3.23094 2.79169 2.730543.51703 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 302 2.39981 3.78637 4.10096 3.27816 3.24279 3.75552 4.072712.01122 3.4022 2.301 2.63105 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 303 2.75206 5.19459 2.29843 1.446723.91789 3.39892 3.69215 3.96001 2.50524 3.48083 4.26301 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 304 2.612613.69779 3.67003 3.30824 2.75096 3.72571 4.03296 2.19251 3.41634 1.865863.03542 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 305 2.61251 4.09277 4.06076 3.47456 2.24559 3.03691 4.012882.35755 3.3562 2.18545 2.938 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 306 2.90848 5.41832 1.29325 1.964674.71447 3.37696 3.7858 4.19593 2.43441 3.70542 4.51235 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 307 2.4578 4.666583.07379 2.54401 4.0584 3.19558 3.8262 3.4527 2.67382 3.09889 3.940772.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 3082.45461 5.01237 2.9391 2.27072 4.30765 3.43681 3.62961 3.75315 2.074562.98146 4.07153 2.68619 4.42226 2.7752 2.73124 3.46339 2.40514 3.724953.29355 2.67734 2.69356 4.24691 0.04727 3.23695 4.97786 0.6378 0.751740.48576 0.9551 309 2.7049 3.94849 3.05443 2.41168 4.30093 3.491623.39525 3.44696 1.62805 3.26986 4.06486 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 310 1.48885 4.62485 3.171312.68033 4.05066 3.04091 3.40451 3.4543 2.65644 3.09295 3.9287 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 311 2.492155.05184 2.76516 2.08438 4.0596 3.41875 3.26516 3.82102 1.93578 3.139124.10278 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 312 2.04528 4.60312 3.18634 2.2123 3.45106 3.50912 3.732762.93956 2.55287 2.83819 3.53467 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 313 3.03702 4.41534 4.68468 4.098692.27125 4.21502 4.4876 2.28531 3.95381 1.15138 2.37605 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 314 2.6119 3.633273.62377 3.37861 2.5622 3.49887 3.47755 2.42549 3.27565 1.96822 3.234852.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.677412.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 3152.28546 5.04925 2.56222 2.25888 4.35344 2.61914 3.61991 3.81188 2.236963.23931 3.77034 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.724943.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.772550.48576 0.9551 316 2.47485 4.83268 2.62191 2.37539 3.74481 3.455853.65933 3.32098 2.36128 2.99025 3.66378 2.68618 4.42225 2.77519 2.731233.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.255524.97786 0.61958 0.77255 0.48576 0.9551 317 2.84582 4.35412 4.780154.22282 3.50189 4.32789 4.74582 1.48701 4.10394 1.99785 2.82026 2.686184.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.693554.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 318 2.622065.18306 3.10741 2.36818 4.56173 3.37691 3.67375 3.96898 1.31727 3.452754.25939 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 319 2.70932 5.37526 1.70108 2.04734 4.68991 3.18323 3.730434.18785 2.21964 3.6687 4.44623 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.0213 4.25552 4.977860.61958 0.77255 0.48576 0.9551 320 3.62982 4.93341 4.85945 4.502710.83157 4.53425 4.0598 2.82803 4.33088 1.89623 3.49824 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.0213 4.25552 4.97786 0.61958 0.77255 0.48576 0.9551 321 3.206084.49457 5.03968 4.48609 3.10426 4.56281 4.97505 1.17306 4.36945 1.806972.84859 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.0213 4.25552 4.97786 0.61958 0.77255 0.485760.9551 322 2.54797 4.44994 3.03683 2.77665 3.20753 3.55489 3.792612.81898 2.6619 2.3977 3.33032 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.09568 4.25552 2.563210.61958 0.77255 0.48576 0.9551 323 2.61023 4.48456 2.85053 2.32024.11292 3.13072 3.63847 3.53709 2.42654 2.86095 3.92985 2.68618 4.422252.77519 2.73123 3.46354 2.40513 3.72494 3.29354 2.67741 2.69355 4.24690.11838 4.18277 2.33941 0.61958 0.77255 0.57559 0.82639 324 2.503463.4029 3.58408 3.01411 3.07703 3.31546 3.36398 2.61861 2.95275 2.263523.32498 2.68618 4.42225 2.77519 2.73123 3.46354 2.40513 3.72494 3.293542.67741 2.69355 4.2469 0.04406 4.08958 3.63611 0.61958 0.77255 0.672070.71468 325 2.42738 4.29616 2.8301 2.29943 4.05943 3.40536 3.610583.4806 2.27795 2.86682 3.88189 2.68618 4.42225 2.77519 2.73123 3.463542.40513 3.72494 3.29354 2.67741 2.69355 4.2469 0.11452 4.07119 2.395240.61958 0.77255 0.68908 0.69723 326 2.42393 4.73588 2.98431 2.438963.97462 3.40392 3.36831 3.25716 2.3385 2.99583 3.81855 2.68591 4.422272.77528 2.73142 3.46372 2.40515 3.72484 3.29372 2.67741 2.69361 4.245320.44569 1.02272 * 1.71775 0.1978 0 * HMM N P Q R S T V W Y COMPO 3.083323.16278 3.14933 2.95336 2.65169 2.88483 2.59999 4.67957 3.50072 2.903852.73746 3.18155 2.8983 2.3788 2.77503 2.98538 4.5849 3.61529 1 2.885263.77642 2.57955 2.68167 2.4586 2.8237 3.21688 5.34464 3.99892  28 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615032 2.70284 3.77638 2.72661 2.75666 2.59095 2.67365 3.19345 5.339533.98699  29 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 3 2.87196 3.77565 2.39793 2.815 2.44517 2.82406 3.30545.41452 4.04333  30 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 4 2.71561 3.83423 2.82061 2.93237 2.148262.68914 3.08385 5.2736 3.70913  31 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 5 2.89654 3.20085 2.820892.74045 2.24849 2.66127 2.99154 4.57462 3.93097  32 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 6 2.921223.28543 2.74721 2.86991 2.27246 2.50614 3.26999 5.40172 3.76473  33 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.615037 2.94258 2.67641 2.76522 2.76329 2.44021 2.84398 3.21693 5.3636 4.01506 34 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 8 2.98477 2.35337 3.01053 3.08784 2.39118 2.58194 2.859015.13831 3.86638  35 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 9 2.88639 2.42343 2.95167 3.03777 2.341 2.687812.89365 5.14493 3.8625  36 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 10 2.68838 3.65725 2.96097 3.043532.36538 2.66847 2.67898 5.0936 3.81758  37 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 11 3.3176 3.712613.16728 3.22162 2.40915 2.54589 2.42603 4.9238 3.68765  38 -- 2.903532.73746 3.18143 2.89807 2.37872 2.7751 2.98525 4.58483 3.6151 12 2.604353.85767 2.72752 2.99753 2.6133 2.98557 3.55449 5.64272 4.23457  42 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.6150313 3.28197 1.87242 3.16811 3.21784 2.47704 2.71377 2.79721 5.136773.88114  43 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 14 2.84662 4.04913 3.23302 3.29664 2.92174 2.763672.78865 4.68319 1.71492  44 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 15 2.84293 3.81574 2.59821 2.848262.62004 2.70016 3.43852 5.51887 4.12974  45 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 16 3.46769 4.017 3.101923.19369 2.71875 2.84504 2.48514 4.78972 2.68075  46 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 17 4.420484.60519 4.20951 4.19678 3.67927 3.35684 2.26569 5.04843 3.94788  47 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.6150318 2.26103 3.84465 2.35167 2.72161 2.6768 2.94597 3.55173 5.597024.20735  48 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 19 4.69285 4.81072 4.46598 4.46288 3.9533 3.516762.04961 5.25471 4.16887  49 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 20 3.16016 3.96901 3.14643 2.911472.83107 2.30885 1.91662 4.96154 3.72256  50 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 21 2.86197 3.357972.54337 2.77232 2.57082 2.85898 2.93104 5.26768 3.57153  51 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 221.09296 3.95591 3.10731 3.39798 2.87684 3.00121 3.76878 5.92587 4.48837 52 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 23 3.41693 1.03535 3.62679 3.62343 2.32477 2.9145 3.3638 5.83674.60374  53 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 24 2.31166 3.97877 2.74169 3.54121 2.96954 3.388334.08478 6.1513 4.62343  54 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 25 3.24288 3.91384 3.49669 3.685312.70827 3.04789 3.53763 5.92879 4.64662  55 -- 2.90341 2.7374 3.181472.89802 2.37888 2.7752 2.98519 4.58478 3.61504 26 3.44969 3.8508 3.611613.6115 1.17415 1.65554 3.28065 5.76893 4.54493  57 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 27 4.38774.58506 4.25882 4.2067 3.62501 3.26538 1.61066 5.10383 3.9568  58 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.6150328 3.68528 4.05006 3.70235 3.66054 2.83531 1.18295 2.47321 5.292334.08047  59 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 29 3.35021 4.08577 2.98303 1.07934 3.01372 3.170123.3723 5.43114 4.22778  60 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 30 2.45453 3.01529 2.68525 2.722312.54764 2.85265 3.13332 5.39381 3.54934  61 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 31 3.37178 3.542443.22706 3.12501 2.30812 2.59326 2.41278 4.87497 3.46753  62 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 322.86463 3.26325 2.75733 2.87797 2.53839 2.52978 3.04053 5.40629 4.04675 63 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 33 3.33944 4.00499 3.33155 3.22215 2.81222 2.72251 2.292364.22539 3.57862  64 -- 2.90349 2.73741 3.18148 2.89803 2.37889 2.775212.9852 4.58479 3.61473 34 3.4287 0.97749 3.55495 3.4574 2.55956 3.022333.29902 5.63307 4.32568  66 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 35 2.89322 3.87297 2.87907 2.765662.32019 2.42136 2.93327 5.22699 3.72907  67 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 36 3.79101 4.208653.68813 3.66223 2.84069 2.38331 1.60478 4.93301 3.73293  68 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 372.80195 2.31234 2.72429 2.9174 2.46453 2.87979 3.10901 5.41707 3.59287 69 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 38 3.06737 2.59171 2.9065 3.00522 2.50811 2.64073 3.02249 5.24733.94336  70 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 39 2.59889 3.49951 2.78908 2.91986 2.22144 2.210883.13513 5.45757 4.09404  71 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 40 2.92785 2.04062 2.83067 2.990062.60202 2.93714 3.38754 5.52462 4.15156  72 -- 2.9034 2.73742 3.181492.89803 2.37889 2.77516 2.98521 4.58479 3.61505 41 2.56911 3.819992.52532 2.90012 2.4293 2.62251 3.50604 5.58015 4.17583  74 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 423.21273 2.46219 3.05297 3.12543 2.6968 2.67956 2.73819 4.42919 3.76111 75 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 43 2.84648 3.85435 2.8428 2.95258 2.33051 2.3019 2.93967 5.279653.95863  76 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 44 3.05565 2.52958 2.90885 2.98981 2.52861 2.440562.98637 5.21884 3.92272  77 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 45 2.90966 3.58965 2.5829 2.873672.51928 2.63962 2.89759 5.38935 4.03054  78 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 46 2.85028 3.832652.84622 2.94179 2.24496 2.60631 2.99409 5.20732 3.90003  79 -- 2.903522.73752 3.18113 2.89783 2.37895 2.77528 2.98514 4.58489 3.61515 473.17979 2.56646 3.02797 2.86056 2.51903 2.75422 2.45651 5.078 3.81545 90 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 48 3.55442 4.02663 3.49007 3.49924 2.50411 2.61715 1.664855.06123 3.84746  91 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 49 3.57365 3.75431 3.45089 3.43793 2.840262.84365 2.03333 4.74524 3.53179  92 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 50 3.36238 3.9572 3.248083.28933 1.67148 2.44429 2.68687 4.99283 3.74412  93 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 51 3.226194.08215 2.87597 2.21843 2.68804 3.24395 3.70322 5.58826 4.37283  94 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.6150352 3.23153 4.23051 3.58785 4.05877 3.38503 3.807 4.31921 6.11573 4.86017 95 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 53 4.85534 4.99585 4.76643 4.71418 4.15094 3.54705 1.33995.68023 4.51296  96 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 54 3.33015 1.65495 3.29233 3.30509 2.579812.02315 2.96046 5.38237 4.13085  97 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 55 4.3535 4.59007 4.17656 4.14553.63988 3.32576 2.02158 5.05775 3.28703  98 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 56 1.17295 3.951223.07197 3.43674 2.63178 3.26288 3.87679 5.99392 4.52402  99 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 572.83035 2.57555 2.61299 2.86292 2.41236 2.77738 3.37675 5.47857 4.10149100 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 58 2.71628 3.82771 2.67225 2.90065 2.18354 2.44744 3.434865.5308 4.14267 101 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 59 2.15446 3.8586 2.77846 2.81135 2.605482.49122 3.43224 5.51626 4.15469 102 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 60 2.28079 3.49617 2.583142.71841 2.39751 2.9076 3.4994 5.56104 4.17059 103 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 61 3.718024.15047 3.69748 3.6497 2.96409 1.21793 2.07913 5.38663 4.15931 104 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.6150362 3.64365 4.14441 3.53448 3.31142 2.79036 2.97282 2.54734 2.091432.86733 105 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 63 4.1695 4.44618 4.03353 3.99646 3.44567 3.168141.71352 4.99906 3.84857 106 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 64 3.80428 4.38334 3.395 0.52404 3.570833.74393 4.02369 5.66284 4.58903 107 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 65 4.85285 4.95471 4.618 4.611434.14693 3.63253 1.87296 5.44859 4.36767 108 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 66 4.32199 4.977634.42893 4.52447 4.07852 4.24492 3.60237 3.68802 1.07985 109 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 673.34726 4.00543 3.1601 2.06091 2.87905 2.90312 2.42108 5.00341 3.77164110 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 68 4.16117 0.34853 4.37081 4.1814 3.52713 3.81811 4.097945.89679 4.96637 111 -- 2.90353 2.73736 3.18153 2.89807 2.37886 2.774532.98525 4.58483 3.61509 69 2.91922 3.62255 2.83304 2.36064 2.328032.49988 2.68205 5.27818 3.96021 122 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 70 2.65101 3.62197 2.625812.75273 2.45298 2.35904 3.42085 5.50488 4.11715 123 -- 2.90348 2.737293.18158 2.89806 2.37889 2.77507 2.98525 4.58489 3.61515 71 2.939382.97669 2.8543 2.96119 2.1677 2.64104 2.96092 5.25328 3.93857 129 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.6150372 2.63519 2.50877 2.85154 2.96849 2.26531 2.86211 2.93905 5.377484.04387 130 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 73 2.7498 3.04638 2.66461 2.87439 2.26911 2.726223.41714 5.51086 4.12418 131 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 74 2.41749 3.82163 2.68319 2.689492.43711 2.60391 3.13389 5.44299 4.07585 132 -- 2.90347 2.7374 3.181472.89801 2.37887 2.7752 2.98519 4.58477 3.61503 75 2.69287 3.512072.79214 2.94362 2.28079 2.15106 3.39419 5.51747 4.13972 134 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 762.68228 3.81886 2.81125 2.92549 2.40989 2.52162 2.74556 5.33759 4.00304135 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 77 2.57096 3.80676 2.59612 2.73634 2.55036 2.76065 3.280375.40117 4.04464 136 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 78 3.33015 4.16424 2.83579 1.61028 3.181733.34459 3.80083 5.56718 4.41071 137 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 79 4.74816 4.91513 4.457474.36509 4.11077 3.78815 2.46198 5.28814 4.11463 138 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 80 4.161170.34853 4.37081 4.1814 3.52713 3.81811 4.09794 5.89679 4.96637 139 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.6150381 4.82118 4.93963 4.64161 4.62074 4.10683 3.57237 1.59668 5.491784.38566 140 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 82 4.8407 4.97703 4.73975 4.69276 4.13067 3.539361.30674 5.63513 4.47926 141 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 83 4.83709 4.97751 4.74739 4.69674.12724 3.53168 1.1862 5.65072 4.4873 142 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 84 4.19844 4.88964.26857 4.34606 3.94889 4.12351 3.62249 3.69146 0.77075 143 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 854.14156 4.57963 4.08777 4.09382 3.59024 3.47095 2.55469 4.13689 2.04898144 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 86 3.79703 4.41988 3.78638 3.48044 3.58665 3.83775 4.007625.08791 3.57711 145 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 87 2.98299 3.87953 3.59841 3.68164 2.376652.97848 3.54566 5.94883 4.67727 146 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 88 3.47564 3.88209 3.799733.83632 2.10584 2.9579 3.53501 5.98367 4.77258 147 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 89 3.502763.91199 3.84058 3.85877 2.36157 3.00557 3.56988 5.98357 4.77492 148 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.6150390 4.17955 4.68714 4.18386 4.21103 3.71458 3.67106 3.05343 3.413452.00158 149 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 91 4.85527 5.00885 4.8066 4.73913 4.1517 3.53479 0.988655.76106 4.56361 150 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 92 4.67795 4.77835 4.41049 4.41953 3.926943.52994 2.08409 5.16441 4.0988 151 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 93 3.80438 4.12233 3.712333.6436 2.91929 2.85989 2.29233 4.64543 2.76597 152 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 94 3.11761 3.5493.00521 2.64849 1.3011 2.96976 3.31349 5.47545 4.16419 153 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 953.55524 3.22825 3.60736 3.60762 2.7151 2.88976 1.92445 5.3993 4.18767154 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 96 2.81245 3.85588 2.86602 3.0359 2.37015 2.54389 3.41127 5.56244.18993 155 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 97 3.00505 3.88038 2.90365 3.0026 2.01908 2.291912.98395 5.20707 3.90669 156 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 98 3.10978 3.17225 2.7977 2.925852.58737 2.51353 2.64506 5.14038 3.85535 157 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 99 3.13135 2.747613.3607 3.37423 2.91179 2.83996 2.40133 4.79664 3.38073 158 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1003.38762 4.08133 3.49624 3.48704 2.98482 2.87964 2.34986 4.68835 3.18047159 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 101 2.6908 3.9553 2.81073 3.09238 2.8756 3.17848 3.6561 5.652994.21696 160 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 102 2.60724 3.81636 2.64977 2.69099 2.55691 2.895313.36501 5.58279 4.17662 161 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 103 3.38285 3.58741 3.24051 3.278612.63286 2.43026 2.56784 4.88648 3.65982 162 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 104 4.41326 4.365924.69189 4.41124 3.37594 3.60566 3.39326 5.76248 4.73113 163 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1052.74926 3.85078 2.82322 2.84007 2.14234 2.62463 3.04237 5.32817 3.99619164 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 106 2.54258 3.8109 2.56533 2.65835 2.39343 2.86594 3.291875.51768 3.78882 165 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 107 3.70816 4.11671 3.58904 3.20257 3.03672.88616 2.17949 4.75507 3.56539 166 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 108 3.50317 3.82571 3.749343.77795 1.83581 2.83574 3.334 5.85879 4.66988 167 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 109 2.923613.82503 2.75196 2.67917 2.27334 2.58943 3.21968 5.47124 4.09825 168 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503110 2.74215 3.81339 2.52207 2.76166 2.55736 2.80216 3.52093 5.576724.17124 169 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 111 3.84841 4.19881 3.74635 3.69846 2.87972 2.820991.98318 4.80657 3.61938 170 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 112 2.76246 2.12847 2.59942 2.912442.6841 2.78697 3.01012 5.36499 4.03038 171 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 113 3.49762 3.927453.52287 3.53044 2.53195 2.4909 2.45812 5.38823 4.17148 172 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1144.82865 4.96286 4.71561 4.67274 4.11561 3.53719 1.31626 5.60027 4.4533173 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 115 4.69765 4.90403 4.6244 4.55898 3.99966 3.48691 0.832385.65286 4.45953 174 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 116 4.10739 4.45305 4.00681 3.97547 3.424433.15654 1.32925 5.13801 3.94577 175 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 117 3.59408 3.96657 3.868683.8086 0.63275 3.06292 3.52165 5.8339 4.58201 176 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 118 3.898443.42511 3.81453 3.76874 3.2146 3.07657 1.24673 5.12779 3.92641 177 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503119 2.82335 3.91826 2.9346 3.26 2.4225 3.19029 3.85503 5.92312 4.45098178 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 120 4.22841 4.76709 4.30182 4.19058 3.93922 4.14006 3.701164.07146 0.50853 179 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 121 3.80428 4.38334 3.395 0.52404 3.570833.74393 4.02369 5.66284 4.58903 180 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 122 3.47596 4.39885 3.84479 3.643.36982 3.35845 2.62714 5.07894 3.73532 181 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 123 3.63145 3.355313.86142 3.82119 2.55547 2.83994 3.09539 5.71638 4.54426 182 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1244.16117 0.34853 4.37081 4.1814 3.52713 3.81811 4.09794 5.89679 4.96637183 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 125 2.97912 4.01507 3.13217 3.28924 2.97306 3.03134 3.732545.91984 4.52338 184 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 126 2.3743 3.93517 2.90644 2.95964 2.834023.11751 3.60861 5.64573 4.25002 185 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 127 3.43138 4.23481 2.916350.88496 3.31059 3.46196 3.87023 5.59281 4.47237 186 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 128 4.702974.84509 4.50218 4.33521 4.10816 3.89905 2.70402 5.33025 4.13656 187 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503129 4.16117 0.34853 4.37081 4.1814 3.52713 3.81811 4.09794 5.896794.96637 188 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 130 3.76814 4.02005 4.06612 3.9693 2.7862 3.091123.25386 5.86855 4.71972 189 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 131 3.39317 3.97023 3.09699 3.283352.7702 2.94935 2.90303 5.35783 4.10375 190 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 132 4.26062 4.919824.34352 4.40766 4.00166 4.1738 3.56508 3.73496 0.65216 191 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1332.80644 3.94187 2.65715 3.36015 2.89171 3.27395 3.9777 6.03531 4.53514192 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 134 3.23153 4.23051 3.58785 4.05877 3.38503 3.807 4.319216.11573 4.86017 193 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 135 3.53526 3.84 3.68065 3.69839 2.40398 2.805782.81028 5.60012 4.41012 194 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 136 3.50926 4.03937 3.37595 3.07922 2.932.61326 1.97187 4.81814 3.60728 195 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 137 2.6935 3.91523 2.926013.04509 2.83069 3.18123 3.85035 5.91761 4.44826 196 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 138 3.558463.84747 3.82739 3.8032 2.28623 2.64101 3.22675 5.84657 4.66418 197 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503139 4.82529 4.94238 4.64265 4.62261 4.11168 3.57699 1.62526 5.491754.38705 198 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 140 2.71222 3.84449 2.79741 2.8297 2.58989 2.72973.20049 5.35755 4.01653 199 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 141 4.00615 4.4762 3.94211 3.893183.43946 3.39323 2.79067 1.19213 2.58759 200 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 142 4.55145 4.785174.49632 4.43629 3.84074 3.37114 1.19156 5.5214 4.3256 201 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1433.34396 4.18615 2.56635 1.23693 3.22682 3.38915 3.89554 5.5991 4.46463202 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 144 2.57803 3.37565 2.65536 2.9027 2.37923 2.42186 3.530775.59818 4.19026 203 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 145 2.77551 3.83157 1.97277 2.86265 2.365632.69173 3.39663 5.49507 4.11945 204 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 146 3.3476 3.95507 2.979163.21123 2.826 2.82194 2.30503 3.90281 3.62419 205 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 147 2.926843.53415 2.79639 2.86504 2.6714 2.7148 2.75975 5.10163 3.81425 206 --2.90313 2.73737 3.18155 2.8982 2.37892 2.77538 2.98509 4.58502 3.61528148 2.86108 3.85204 2.83366 2.96774 2.41809 2.89927 3.27361 5.432884.08203 215 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 149 2.827 3.9295 2.947 2.84094 2.85851 3.2174 3.89155.95037 4.47796 216 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 150 2.86973 2.25357 2.54389 2.97094 2.694592.85852 3.58537 5.65728 4.24341 217 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 151 4.04957 4.49307 3.988273.80764 3.47484 3.51075 2.50914 1.00434 2.78975 218 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 152 4.723094.85061 4.4628 4.48098 4.02107 3.58102 1.88462 5.28186 4.19755 219 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503153 2.83966 3.84698 2.74318 2.31801 2.40296 2.72285 3.24603 5.475044.12399 220 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 154 2.47841 3.84817 2.80209 2.91069 2.48506 3.012693.6648 5.72456 4.28964 221 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 155 3.50527 4.14049 3.37848 3.219293.03504 3.04768 2.76118 4.40663 1.67141 222 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 156 3.46051 3.928533.45577 3.475 2.70269 2.85377 2.05286 5.27525 4.05253 223 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1573.18455 4.1833 3.53233 3.99216 3.32886 3.74622 4.24748 6.06325 4.79998224 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 158 3.73503 3.52921 3.62282 3.59553 3.07632 2.93642 2.326184.62538 2.91699 225 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 159 2.77504 3.86695 3.45173 3.48253 0.922772.95732 3.45348 5.76287 4.44925 226 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 160 2.37252 3.96408 2.767891.79599 2.85932 2.93721 3.628 5.55554 4.27472 227 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 161 3.865184.15703 3.87085 3.80476 2.84726 2.8073 1.56074 5.09092 3.89445 228 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503162 4.22347 4.78698 4.25797 4.30799 3.83111 3.85336 2.8556 3.834911.53446 229 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 163 4.80849 4.90642 4.54591 4.55791 4.09365 3.614911.88885 5.36168 4.31003 230 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 164 3.98729 4.34003 3.84639 3.798013.30327 3.11266 2.30468 4.9072 3.39865 231 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 165 4.13001 4.334124.44637 4.32905 3.39874 3.72363 4.18538 5.99711 5.14174 232 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1663.03569 3.90075 3.00573 3.09204 1.84856 2.49828 2.87913 5.14368 3.86879233 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 167 3.02832 3.9191 3.05809 3.18797 1.20559 3.05132 3.451835.56968 4.18219 234 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 168 2.63706 3.84577 3.36963 3.44049 2.095992.60546 3.27361 5.66066 4.40726 235 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 169 4.13001 4.33412 4.446374.32905 3.39874 3.72363 4.18538 5.99711 5.14174 236 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 170 3.53373.86418 3.82864 3.85176 2.57111 2.90314 3.40139 5.93785 4.76384 237 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503171 1.14369 3.9346 3.26019 3.30428 2.7607 2.3136 3.4609 5.74715 4.40951238 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 172 4.79062 4.91387 4.55933 4.54474 4.08911 3.59769 2.026225.40471 4.3178 239 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 173 3.55254 3.92217 3.62831 3.63291 2.43232.56229 2.06166 5.42804 4.22422 240 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 174 4.25836 4.84403 4.303084.36634 3.90761 3.95172 3.28836 3.79313 1.01641 241 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 175 2.326343.86247 2.48809 2.69167 2.68159 2.88789 3.22597 5.30912 3.57131 242 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503176 3.70806 4.13653 3.67338 3.66534 2.96651 2.45344 1.58869 5.238134.02633 243 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 177 3.09386 3.8353 3.58054 3.65727 2.38841 2.868863.38279 5.84237 4.61572 244 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 178 4.84385 4.93546 4.49536 4.526854.16262 3.72765 2.26283 5.32102 4.30308 245 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 179 2.95959 3.908522.73145 1.71155 2.75236 2.9299 3.19092 4.55388 4.02765 246 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1803.45764 3.96581 3.39827 3.4257 2.32623 2.68907 2.05848 5.15342 3.93086247 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 181 3.49135 4.01152 3.21082 3.38793 2.78551 2.85216 2.315154.88732 3.67459 248 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 182 2.59984 3.82397 2.74555 2.89028 2.570942.89663 3.30162 5.54537 4.15131 249 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 183 3.10273 3.93899 3.067932.98479 2.64808 2.75893 2.46463 5.01643 3.76148 250 -- 2.90353 2.737463.18153 2.898 2.37887 2.77526 2.98489 4.58483 3.61509 184 2.77556 3.95342.84007 3.52097 2.77605 3.36463 4.05969 6.13946 4.61155 253 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1854.23309 4.51695 4.05418 4.04354 3.54514 2.79737 2.05308 5.05967 3.93294254 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 186 2.76217 3.82276 2.75088 2.91665 2.09891 2.81435 3.543075.61098 4.19982 255 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 187 3.18428 1.33641 3.21308 3.22378 2.700372.97541 3.35691 5.64772 4.36442 256 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 188 4.44297 4.64734 4.297874.25943 3.70513 3.32735 1.63114 5.19097 4.06177 257 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 189 2.972833.8563 2.61019 2.44541 2.68096 2.77295 3.34473 5.43447 4.09447 258 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503190 4.84266 4.98596 4.71018 4.66313 4.14762 3.57919 1.64071 5.619824.46683 259 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 191 2.96886 3.84271 2.50606 2.23991 2.43867 2.756053.10587 5.38164 4.03615 260 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 192 4.13001 4.33412 4.44637 4.329053.39874 3.72363 4.18538 5.99711 5.14174 261 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 193 4.42883 4.641244.25288 4.22803 3.70233 3.35877 1.85988 5.16553 4.05112 262 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 1944.32006 4.60954 4.25312 4.18473 3.60893 3.2602 1.47614 5.33681 4.13282263 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 195 4.87099 4.92537 4.50327 4.55713 4.16435 3.72988 2.500695.24999 4.22787 264 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 196 2.01295 3.83746 2.62923 2.78951 2.651082.88278 3.32791 5.44105 3.78625 265 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 197 3.06148 3.91993 1.560252.78935 2.76841 2.9663 3.1073 5.3531 4.0176 266 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 198 3.477591.07138 3.55227 3.47022 2.74934 2.99572 3.02021 5.57559 4.30789 267 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503199 3.85272 4.36901 3.78968 3.79415 3.06756 3.25556 2.7829 4.116821.90503 268 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 200 4.48245 4.84946 4.37958 4.39217 3.97301 3.819472.78356 4.34976 2.73477 269 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 201 3.43781 3.83194 3.68626 3.735441.79077 2.55653 3.42345 5.92287 4.71349 270 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 202 3.61161 3.98053.95199 3.93802 2.75595 3.10087 3.59477 5.99348 4.85068 271 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2033.29571 3.96869 3.14178 3.19598 2.676 2.43226 1.94094 4.97522 3.73231272 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 204 2.8777 3.82831 2.55963 2.8794 2.64761 2.72971 3.545635.60351 4.19684 273 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 205 3.17947 3.63101 2.88986 1.15319 2.819683.18288 3.59806 5.56228 4.322 274 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 206 3.45519 3.9076 3.453033.46688 2.02051 1.35458 2.84596 5.3295 4.10639 275 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 207 2.875253.0584 2.5371 2.73481 2.63606 2.90857 3.53788 5.59647 4.18833 276 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503208 3.49912 3.84518 3.75548 3.75749 0.86738 2.87074 3.35269 5.891924.68098 277 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 209 2.90712 4.0313 3.1536 3.5168 3.04275 3.44139 4.031086.0823 4.63717 278 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 210 3.65396 4.10439 3.53411 3.51415 2.892272.72032 2.13945 4.79947 3.59983 279 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 211 2.88129 4.01724 2.805961.40204 2.9345 3.14584 3.64213 5.55856 4.30545 280 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 212 2.9443.99632 3.21207 3.07928 2.47706 2.87363 2.62157 4.89026 3.40426 281 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503213 3.29944 3.96475 3.14626 2.99158 2.82519 2.84993 2.19138 4.949483.70917 282 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 214 1.73014 3.88825 2.89428 3.1513 2.65166 2.735623.67772 5.77869 4.34804 283 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 215 2.97097 4.05638 3.29801 3.80043.07468 3.49776 4.13871 6.17808 4.76353 284 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 216 2.92666 2.913352.44294 2.35575 2.4315 2.87572 3.38109 5.47602 3.88189 285 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2173.44356 4.05419 3.44165 3.29071 2.84849 2.63023 1.70902 4.7806 3.57608286 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 218 4.30461 4.50902 4.14108 4.10077 3.54181 3.23769 2.152234.98317 3.85773 287 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 219 3.38069 1.15025 3.43381 3.35959 2.465042.97792 3.17622 5.51871 4.21657 288 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 220 3.07721 3.0092 3.2694 3.26372.66456 2.89078 2.57005 5.02262 3.78267 289 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 221 3.21588 2.526132.94699 3.12992 2.58578 2.75312 2.67351 5.03407 3.77642 290 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2223.14395 3.98051 3.25292 3.28556 2.76033 2.75863 1.98205 4.87558 3.67659291 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 223 2.18317 3.90854 3.02104 3.10187 2.49669 2.34772 2.746535.12823 3.85713 292 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 224 2.88695 4.02477 3.15442 3.57919 3.031813.43913 4.05741 5.96487 4.4489 293 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 225 3.65478 4.11341 3.521763.10264 3.02688 2.90444 2.04871 4.80614 3.61104 294 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 226 4.02034.32364 3.88904 3.84436 3.29242 2.84047 2.27852 4.88107 3.71993 295 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503227 4.07041 4.51704 4.01665 3.82098 3.5109 3.56822 2.6592 0.939312.74344 296 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 228 2.74043 3.84877 2.69295 2.98051 2.17151 2.983953.6221 5.68425 4.26026 297 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 229 4.06974 4.47111 3.90435 3.899413.46749 3.28148 2.36925 4.99959 3.75961 298 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 230 3.53126 3.855273.6436 3.66186 1.67824 2.62186 2.8189 5.51449 4.31994 299 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2314.70297 4.84509 4.50218 4.33521 4.10816 3.89905 2.70402 5.33025 4.13656300 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 232 4.16117 0.34853 4.37081 4.1814 3.52713 3.81811 4.097945.89679 4.96637 301 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 233 3.08763 3.92642 3.00578 3.08654 2.769672.86943 1.99402 5.11237 3.8387 302 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 234 3.20718 3.91966 3.47943.68802 2.53414 3.07678 3.60843 5.95082 4.64243 303 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 235 3.571073.95413 3.58438 3.59272 2.13812 2.56649 1.71302 5.18442 3.98287 304 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503236 2.5037 3.98042 3.12571 3.60425 2.95902 3.10524 4.01731 6.145624.63274 305 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 237 3.33142 4.14725 2.64027 0.99486 3.1434 3.086563.69625 5.55454 4.37587 306 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 238 2.69652 3.98805 3.1623 3.676032.71784 3.40211 4.06257 6.17855 4.65259 307 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 239 3.79703 4.419883.78638 3.48044 3.58665 3.83775 4.00762 5.08791 3.57711 308 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2402.98933 3.89375 2.8645 2.66573 2.74007 2.97191 2.71063 5.48831 4.14512309 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 241 4.08871 4.64221 4.09598 4.12045 3.6343 3.65785 3.083983.91121 0.85287 310 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 242 3.59529 3.94484 3.63802 3.62731 1.722142.84419 2.7186 5.16428 3.9425 311 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 243 0.92123 4.01695 3.21623.51804 2.98116 3.35957 3.89955 5.94846 4.50832 312 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 244 3.637290.93137 3.64824 3.49941 3.03624 3.24099 3.122 5.44029 4.14682 313 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503245 3.46225 4.03364 3.31958 3.21658 2.91809 2.41827 2.33006 4.869523.65313 314 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 246 3.13012 3.90333 2.86653 2.93774 2.54333 2.764192.40077 5.12998 3.84971 315 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 247 2.65165 3.81945 2.74001 2.735572.50587 2.8631 3.23909 5.46696 3.81282 316 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 248 2.55415 3.611782.59243 2.78996 2.55147 2.79948 3.39944 5.49285 4.10988 317 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2492.66634 3.41531 2.79728 2.91738 2.46457 2.86855 3.23119 4.22213 4.03642318 -- 2.90347 2.7374 3.18147 2.89801 2.37887 2.7752 2.98511 4.584773.61503 250 2.97532 3.41289 2.74765 2.68978 2.28116 2.7845 2.801955.17138 3.86328 320 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 251 3.00997 3.82478 2.74314 2.7896 2.459992.80588 2.53965 5.1557 3.85547 321 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 252 2.8122 3.7905 2.6251 2.621352.47224 2.83958 3.35964 5.4551 3.74485 322 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 253 2.83295 3.589922.78824 3.05358 2.7367 2.84284 2.83694 5.09255 3.3308 323 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2542.77944 3.81922 2.74459 2.90152 2.56399 2.90986 3.49974 5.57629 4.17314324 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 255 2.54247 3.84427 2.69556 2.5689 2.65794 2.87639 3.280825.40665 3.84179 325 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 256 4.74398 4.88662 4.61038 4.5748 4.019583.49274 1.40449 5.47814 4.34912 326 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 257 3.05039 3.92287 2.812082.10241 2.77657 2.87807 3.47322 5.51568 4.20875 327 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 258 2.914383.83401 2.65614 1.93814 2.5552 2.90262 3.21183 5.53655 4.15574 328 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503259 3.8897 4.28429 3.47181 3.69831 3.24204 3.07326 2.32841 4.87153.68322 329 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 260 2.94878 2.34022 2.61667 2.58698 2.68977 2.82413.4139 5.50433 4.14518 330 -- 2.9035 2.73743 3.1815 2.89804 2.37892.77523 2.98522 4.58104 3.61507 261 2.91846 3.26698 2.66368 2.062612.6113 2.83052 3.32774 4.33587 4.08982 332 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 262 4.10273 4.389254.04113 3.98024 3.33621 2.92421 1.52938 5.06809 3.87243 333 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2634.17895 4.46218 3.9882 3.50849 3.48131 3.24376 2.38294 4.93975 3.79486334 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 264 4.66922 4.86428 4.57938 4.53513 3.96412 3.45481 0.92447 5.524.33392 335 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 265 2.88487 3.9778 3.1923 2.69826 2.68678 2.469722.38116 4.913 3.4294 336 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 266 3.30168 3.9531 3.18156 3.212712.65066 2.87878 2.58741 5.07124 3.40935 337 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 267 2.69055 3.966443.14739 2.93636 2.71216 2.85395 2.65451 4.9359 2.5173 338 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2682.71543 3.82018 2.73692 2.61288 2.51777 2.88583 3.44807 5.53194 4.14028339 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 269 2.92877 3.91898 2.93968 2.62672 2.80567 3.09658 3.582885.70003 4.31784 340 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 270 3.23153 4.23051 3.58785 4.05877 3.385033.807 4.31921 6.11573 4.86017 341 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 271 3.4468 1.30454 3.394933.31617 2.82687 2.80687 2.7958 5.36469 4.09397 342 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 272 3.755393.48523 3.62504 3.59527 2.9501 2.97448 2.17806 4.87076 3.68552 343 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503273 3.74609 4.1125 3.64019 3.18714 3.03682 2.86575 1.74391 4.689963.25168 344 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 274 3.23153 4.23051 3.58785 4.05877 3.38503 3.8074.31921 6.11573 4.86017 345 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 275 3.389 4.20134 2.87837 0.97353 3.24593.39214 3.76839 5.5477 4.40218 346 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 276 2.91073 3.89154 1.557692.88255 2.46304 2.69593 3.29399 5.45123 4.12584 347 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 277 3.122663.91936 2.70995 2.08216 2.76398 2.89886 2.6136 5.20313 3.92266 348 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503278 2.87207 3.8426 2.77641 2.74598 2.67595 2.72565 3.56541 5.635594.22407 349 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 279 4.552 4.74032 4.33421 4.338 3.85212 3.52054 1.977124.97337 3.70068 350 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 280 4.04311 4.38632 4.03676 3.98298 3.265263.15902 1.13278 5.42087 4.20578 351 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 281 2.57098 3.87941 2.747652.05076 2.72001 2.84902 3.57584 5.58202 4.22358 352 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 282 3.834354.17303 3.71749 3.44045 3.11301 2.92441 2.26398 4.03334 3.56339 353 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503283 4.79625 4.9058 4.45289 4.46832 4.1156 3.71584 2.31132 5.292244.26251 354 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 284 2.94202 3.84477 2.63707 2.69451 2.66183 2.901373.10568 5.48944 4.12644 355 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 285 2.79507 3.82214 2.73762 2.794512.56577 2.69653 3.46822 5.54614 4.15266 356 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 286 2.60584 3.853912.76764 2.15057 2.53072 2.8974 3.33988 5.44131 4.09469 357 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2874.13001 4.33412 4.44637 4.32905 3.39874 3.72363 4.18538 5.99711 5.14174358 -- 2.90348 2.73741 3.18148 2.89802 2.37888 2.77521 2.9852 4.584783.61504 288 4.36241 4.66366 4.31173 4.24713 3.65397 3.0981 0.91295.51418 4.30654 362 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 289 2.6299 3.81865 2.38047 2.68704 2.63072.90437 3.55224 5.60138 4.1915 363 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 290 4.39667 4.66484 4.385064.27796 3.62653 3.35899 0.81078 5.60905 4.38851 364 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 291 3.069773.89719 2.91543 3.00163 2.73196 2.64931 1.99781 5.25273 3.94904 365 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503292 2.98093 3.84902 2.81133 2.63405 2.56721 2.7822 2.95326 5.384684.04366 366 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 293 2.80617 3.82557 2.65643 2.28622 2.51721 2.877273.16808 5.49435 4.11885 367 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 294 3.89641 4.23778 3.78696 3.741513.01456 2.99923 2.27151 4.64053 3.04737 368 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 295 2.78638 3.817172.66238 2.75489 2.46889 2.62557 2.90962 5.48798 3.65338 369 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 2962.8779 3.83106 2.65698 2.80591 2.65298 2.92588 3.01843 5.60076 4.19476370 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 297 2.94169 3.85121 2.8143 2.95598 2.67677 2.90977 3.150865.45641 3.65663 371 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 298 2.99871 3.89494 3.04139 3.23675 2.496533.02301 3.24829 5.6479 4.28587 372 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 299 3.6964 4.10981 3.591713.55629 3.02793 2.88812 2.23928 4.5871 2.60401 373 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 300 3.797034.41988 3.78638 3.48044 3.58665 3.83775 4.00762 5.08791 3.57711 374 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503301 3.04541 3.89626 3.12507 3.18587 2.58664 2.85626 2.80437 5.241993.97819 375 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 302 3.73443 4.12588 3.62914 3.59162 2.7763 2.880731.80521 4.76515 3.57129 376 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 303 2.87458 3.86163 2.65692 3.010542.71131 2.79001 3.56132 5.65054 4.23924 377 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 304 3.72968 4.093843.62657 3.57749 3.01518 2.7385 2.14422 4.68749 2.9652 378 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 3053.68774 4.08206 3.57886 3.40275 2.763 2.84466 2.10693 4.7005 3.22265 379-- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 306 2.84039 3.91812 2.92771 3.21026 2.83117 3.16936 3.371295.8699 4.41766 380 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 307 3.10827 1.72208 2.89443 3.09457 2.607552.91108 2.79239 5.37431 4.0777 381 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 308 2.65587 3.83289 2.673032.74306 2.10359 2.81032 3.37697 5.47497 4.10635 382 -- 2.90347 2.73743.18147 2.89802 2.37888 2.7752 2.98519 4.58478 3.61504 309 2.889173.87593 2.57634 2.42638 2.70625 2.92664 3.3701 5.44806 4.11455 385 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503310 3.13176 3.8772 3.0175 2.85652 2.48078 2.68303 3.11756 5.36133 4.0705386 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 311 2.81549 3.81291 2.64312 2.84448 2.54176 2.79728 3.270955.50626 4.11911 387 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 312 3.00338 3.70292 2.94366 3.03676 2.64182.72316 2.70038 5.14794 3.86175 388 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 313 4.26075 4.49747 4.078094.05218 3.32305 3.26145 2.40478 4.9215 3.7577 389 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 314 3.629344.06528 3.5133 3.49234 2.97307 2.6411 2.37715 4.72089 2.69149 390 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503315 2.67215 3.81757 2.66453 2.86274 2.62285 2.76226 3.41816 5.507824.12161 391 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 316 2.89859 3.84575 2.63854 2.83459 2.38629 2.770762.85238 5.33411 3.46915 392 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 317 4.3899 4.634 4.30688 4.24796 3.662853.06959 1.30038 5.27231 4.09455 393 -- 2.90347 2.73739 3.18146 2.898012.37887 2.77519 2.98518 4.58477 3.61503 318 3.06474 3.96574 2.795842.24886 2.85305 3.08196 3.60033 5.56411 4.2719 394 -- 2.90347 2.737393.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 319 2.268053.88228 2.85632 3.11139 2.65155 3.08567 3.75366 5.81284 4.36273 395 --2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503320 4.42942 4.84922 4.38092 4.38584 3.958 3.88041 2.89332 4.221252.56544 396 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.985184.58477 3.61503 321 4.65005 4.80648 4.5078 4.47436 3.91515 3.442351.5474 5.35734 4.22496 397 -- 2.90347 2.73739 3.18146 2.89801 2.378872.77519 2.98518 4.58477 3.61503 322 3.05966 3.93739 3.06446 2.733732.35106 2.85001 2.73416 4.38126 3.13613 398 -- 2.90347 2.73739 3.181462.89801 2.37887 2.77519 2.98518 4.58477 3.61503 323 2.84932 3.822552.78012 2.70216 2.03782 2.58111 3.19901 5.35527 4.01084 399 -- 2.903472.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.58477 3.61503 3243.37707 3.95525 3.23876 3.26915 2.5015 2.4114 2.43034 4.80927 3.5886 400-- 2.90347 2.73739 3.18146 2.89801 2.37887 2.77519 2.98518 4.584773.61503 325 2.93089 3.32053 2.62015 2.86785 2.354 2.73771 3.039095.30984 3.96862 401 -- 2.90347 2.73739 3.18146 2.89801 2.37887 2.775192.98518 4.58477 3.61503 326 2.69886 3.79927 2.6598 2.74064 2.247122.81046 2.87558 5.24658 3.92536 402 -- 2.90355 2.7376 3.18172 2.898342.37864 2.77524 2.98456 4.58511 3.61522

What is claimed is:
 1. A method of increasing in a crop plant at leastone phenotype selected from the group consisting of: triple stresstolerance, drought stress tolerance, nitrogen stress tolerance, osmoticstress tolerance, ABA response, tiller number, yield and biomass, themethod comprising increasing the expression of a carboxyl esterase inthe crop plant.
 2. The method of claim 1, wherein the crop plant ismaize and the carboxyl esterase is a plant carboxyl esterase.
 3. Themethod of claim 1, wherein the carboxyl esterase has at least 80%sequence identity, when compared to SEQ ID NO:18, 39, 43, 45, 47, 49,51,55, 59, 61, 64, 65, 66, 95, 97, 101, 103, 107, 111, 113, 117, 119,121, 123, 127, 129, 130, 131, 132, 135, 627 or
 628. 4. (canceled)
 5. Aplant comprising in its genome a recombinant DNA construct comprising apolynucleotide operably linked to at least one heterologous regulatoryelement, wherein said polynucleotide encodes a polypeptide having anamino acid sequence of at least 80% sequence identity, when compared toSEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97,101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132,135, 627 or 628, and wherein said plant exhibits an increase in yield,biomass, or both, when compared to a control plant not comprising saidrecombinant DNA construct.
 6. The plant of claim 5, wherein said plantexhibits said increase in yield, biomass, or both when compared, underwater limiting conditions, to said control plant not comprising saidrecombinant DNA construct.
 7. The plant of claim 5, wherein said plantis selected from the group consisting of: Arabidopsis, maize, soybean,sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley,millet, sugar cane and switchgrass.
 8. Seed of the plant of claim 5,wherein said seed comprises in its genome a recombinant DNA constructcomprising a polynucleotide operably linked to at least one heterologousregulatory element, wherein said polynucleotide encodes a polypeptidehaving an amino acid sequence of at least 80% sequence identity, whencompared to SEQ ID NO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61, 64, 65,66, 95, 97, 101, 103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130,131, 132, 135, 627 or 628, and wherein a plant produced from said seedexhibits an increase in at least one phenotype selected from the groupconsisting of: drought stress tolerance, triple stress tolerance,osmotic stress tolerance, nitrogen stress tolerance, tiller number,yield and biomass, when compared to a control plant not comprising saidrecombinant DNA construct. 9-14. (canceled)
 15. An isolatedpolynucleotide comprising: (a) a nucleotide sequence encoding apolypeptide with stress tolerance activity, wherein the stress isselected from a group consisting of drought stress, triple stress,nitrogen stress and osmotic stress, and wherein the polypeptide has anamino acid sequence of at least 95% sequence identity when compared toSEQ ID NO:18, 39, 43, 45, 47, 49, 51,55, 59, 61,64, 65, 66, 95, 97, 101,103, 107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135,627 or 628; or (b) the full complement of the nucleotide sequence of(a), wherein the nucleotide is operably linked to a heterologousregulatory element.
 16. The polynucleotide of claim 15, wherein theamino acid sequence of the polypeptide comprises SEQ ID NO:18, 39, 43,45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101, 103, 107, 111, 113,117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or
 628. 17. Thepolynucleotide of claim 15 wherein the nucleotide sequence comprises SEQID NO:16, 17, 19, 38, 42, 44, 46, 48, 50, 54, 58, 60, 62, 63, 94, 96,100, 102, 106, 110, 112, 116, 118, 120 or
 122. 18. (canceled)
 19. Aplant comprising in its genome an endogenous polynucleotide operablylinked to at least one heterologous regulatory element, wherein saidendogenous polynucleotide encodes a polypeptide having an amino acidsequence of at least 80% sequence identity, when compared to SEQ IDNO:18, 39, 43, 45, 47, 49, 51, 55, 59, 61, 64, 65, 66, 95, 97, 101, 103,107, 111, 113, 117, 119, 121, 123, 127, 129, 130, 131, 132, 135, 627 or628, and wherein said plant exhibits at least one phenotype selectedfrom the group consisting of increased triple stress tolerance,increased drought stress tolerance, increased nitrogen stress tolerance,increased osmotic stress tolerance, altered ABA response, altered rootarchitecture, increased tiller number, when compared to a control plantnot comprising the heterologous regulatory element. 20-28. (canceled)29. The plant of claim 19, wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 95% sequenceidentity, when compared to SEQ ID NO:18.
 30. The plant of claim 19 ismaize plant.
 31. The plant of claim 19, wherein the heterologous nucleicacid is a constitutive plant promoter.